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The Life of Eukaryotic mRNA: Localization, Translation, and Degradation

Part 1: mRNA Localization, Translation and Degradation

00:00:00.00 Hello, I'm Roy Parker. I'm a professor at the University of Arizona,
00:00:04.14 and an investigator at the Howard Hughes Medical Institute. Today I'd like to talk to you
00:00:08.09 about the life of the eukaryotic mRNA. Eukaryotic mRNAs are produced
00:00:12.22 in the nucleus, and then exported to the cytoplasm, where they can be localized to specific regions,
00:00:17.27 translated to produce protein, and ultimately degraded.
00:00:21.26 In my talks today, I'll cover two areas. In the first lecture,
00:00:26.16 I'll talk about the mechanisms that control
00:00:29.01 localization, translation, and degradation. And in the second part of my talk,
00:00:34.02 I'll talk about structures in the cells that we've been studying, referred to as P-bodies,
00:00:37.28 and what they tell us about a dynamic transition mRNAs undergo, we refer to
00:00:42.22 as the mRNA cycle.
00:00:45.04 Before beginning my talk about the basic mechanisms of translation and
00:00:51.12 localization, and degradation,
00:00:52.20 I want to say a few words about how mRNAs are produced in eukaryotic cells.
00:00:56.24 mRNAs are produced in the nucleus, by transcription, followed by RNA processing.
00:01:02.18 Which includes splicing, and the addition of a three prime poly-A tail.
00:01:06.18 One of the interesting features about eukaryotic mRNAs
00:01:10.09 is that there can be a wide of diversity of events which alter the form of the mRNA
00:01:15.26 you can have alternative transcription starts,
00:01:17.19 alternative splicing, and alternative polyadenylation. And this leads to a wide diversity of
00:01:23.17 transcripts being produced from a single gene
00:01:24.25 , which can influence the nature of the proteins produced, as well as the features of the mRNA
00:01:29.27 which control its localization, translation and degradation.
00:01:37.02 During the process of RNA biogenesis in the nucleus, a number of factors begin to associate
00:01:42.16 with the mRNA. These include RNA binding proteins, which bind to the transcript,
00:01:46.22 but in addition there are certain complexes which are added
00:01:50.07 during the process of RNA processing. For example, the process of splicing,
00:01:54.25 where introns are removed, can deposit a
00:01:56.29 complex of proteins, referred to as the exon junction complex
00:02:00.27 at the site were the intron was removed. Many of these factors then travel out to
00:02:05.25 the cytoplasm with the mRNA, and can affect its localization,
00:02:09.05 translation, and degradation. And we will discuss some of these later on in the talk.
00:02:15.12 Now, once in the cytoplasm, mRNAs can be localized to specific regions.
00:02:19.28 For example, this is an image of a crawling fibroblast, and the fibroblast could be crawling
00:02:25.27 towards me across the screen. Within the fibroblast, this red structure
00:02:30.27 is actually the beta-actin mRNA. And you can see it is localized to this leading
00:02:36.00 edge of the fibroblast cell. And that is because actin filaments are assembling at those sites
00:02:41.26 and so the cell needs newly synthesized actin, preferentially in this region of the cell.
00:02:50.07 So this illustrates one of the reasons that mRNAs are localized in
00:02:53.21 the eukaryotic cell, is to allow local production of protein. But there are other examples
00:02:57.21 where we need to localize mRNAs for other reasons.
00:03:00.26 For example, in neurons, the localization of mRNAs to specific synapses
00:03:05.12 allows the rapid and local response to various stimuli.
00:03:09.08 Localizing RNAs can also limit inappropriate interaction and can facilitate
00:03:14.23 the assembly of protein. For example, by localizing mRNAs to some membrane compartments
00:03:19.27 proteins can be transported across that membrane during their biogenesis
00:03:24.01 more efficiently.
00:03:28.01 Now one of the other interesting events in the last five years
00:03:31.28 has been that we learned that many mRNAs are localized in eukaryotic cells.
00:03:36.12 This particular slide illustrates an experiment done in Drosophila embryos
00:03:40.21 where about 3,000 mRNAs were examined for their localization within the embryo.
00:03:45.22 Now what you can see here is that there is a large number of
00:03:49.06 diverse patterns. That these mRNAs are not just distributed uniformly across this cell.
00:03:53.25 In fact, of those 3,000 mRNAs, about 71% were actually localized, suggesting that
00:04:00.05 the vast number of mRNAs are localized to specific regions.
00:04:04.22 This is true even in very simple cells.
00:04:07.21 For example, if we look at a yeast cell, which is considered a relatively simple eukaryotic cell,
00:04:13.12 we know that about 500 mRNAs are targeted to the surface of the mitochondria.
00:04:17.21 We know that about another 700 or so are targeted to the
00:04:20.19 surface of the ER. And we even know that about 20 are targeted to the very tip of the growing cell,
00:04:25.18 The so called bud tip. And so even in this organism, looking at a few compartments
00:04:31.09 we know of over 1,000 mRNAs that are targeted to specific regions.
00:04:35.15 And so the conclusion that we have to draw from this is that there is lots of localization
00:04:39.25 of mRNAs in the eukaryotic cell. And that the mechanisms which do that and the specificity
00:04:43.29 are an important part of the control of mRNAS in eukaryotic cells.
00:04:54.22 So an important question then becomes where does the specificity for localization of mRNAs
00:05:00.13 come from? And I want to use the example of Yeast ASH1
00:05:04.21 mRNA, which is targeted to the bud tip as an example of that.
00:05:08.23 And the basic principal is that the specificity for transporting this RNA
00:05:13.26 out here to the bud tip is found in RNA binding proteins which bind to the RNA
00:05:19.12 and then attach that to cytoskeletal motors. For example, in this particular RNA
00:05:24.29 the She2 RNA binds to the message, and then interacts with a motor complex
00:05:30.00 which then moves along actin filaments, so that it reaches the bud tip.
00:05:35.06 This is a general principle that RNA binding proteins bind to localized RNAs
00:05:40.12 and then target them to specific regions. In many cases that is by interacting with cytoskeleton motors,
00:05:45.18 but in some cases it's by interacting with anchors, which are localized to certain parts of the cell
00:05:50.09 that tether the RNA to that vicinity.
00:05:56.03 mRNAs can also be localized by selective degradation of the non-localized pool.
00:05:59.28 So for example, mRNAs can start out distributed throughout the cell,
00:06:05.01 but then RNAs that are not in a specific location can be preferentially degraded.
00:06:09.24 One example of this is the nanos mRNA which is localized to the posterior tip of
00:06:14.14 the Drosophila embryo. It's distributed throughout the cell
00:06:17.22 but then degraded in the regions that are not the posterior pole.
00:06:24.03 Another important general principle of RNA transport
00:06:26.22 is that mRNAs are translationally repressed during the transport process.
00:06:30.21 This is important for two reasons.
00:06:33.11 First, by repressing the mRNA
00:06:36.00 so it is not translated prior to localization, you target the production of the protein
00:06:40.26 to a specific region of the cell
00:06:42.15 where you would like to produce the protein.
00:06:44.03 The second reason this is important, is that by repressing the translation of the mRNA,
00:06:48.18 you limit the interaction of the ribosome with the mRNA, and this decreases
00:06:53.15 the total molecular mass of the complex that needs to be moved through the cell.
00:06:57.15 And this presumably greatly facilitates the transport of mRNAs
00:07:01.11 to distal regions. In the case of the Ash1 mRNA, we can see an example of how this occurs.
00:07:09.07 Two RNA binding proteins, one Puf6, and one Khd1, bind to the RNA
00:07:15.22 and then repress the loading of ribosome subunits. In fact, there are two
00:07:21.02 mechanisms by which translation is repressed, which illustrates how important it is to keep
00:07:25.00 these localized mRNAs from being translated prematurely before they reach their proper location.
00:07:32.20 Now, if mRNAs are repressed during their transport
00:07:35.20 an important second principle is that their repression must be relieved once they reach their proper site.
00:07:43.21 And for example, in the case of the Ash1 mRNA, once the mRNA reaches this bud tip
00:07:48.26 region, there are localized kinases, which are present only in this region of the cell,
00:07:52.26 which phosphorylate these RNA binding proteins, they are released from the mRNA
00:07:57.22 and the mRNA can then enter translation. So the two general principles there that
00:08:02.23 are to be remembered, mRNAs are translationaly repressed
00:08:05.01 during transport, and that once they reach their specific sub-cellular location,
00:08:08.21 that repression needs to be relieved, and in some cases by previously localized kinases.
00:08:18.00 Now, once an mRNA reaches its proper place, it needs to enter translation.
00:08:26.01 Translation is a complex process.
00:08:28.11 And it has three different phases.
00:08:30.12 Translation initiates in what is called the initiation phase, where ribosomes load on the message.
00:08:35.13 Followed by elongation where the poly peptide chain is produced
00:08:38.22 where the ribosome moves along the mRNA, translating each of the codons
00:08:43.08 followed by a termination phase, where the ribosome recognizes the stop codon
00:08:47.25 , releases the newly produced protein,
00:08:50.07 and the ribosome subunits disassemble.
00:08:53.07 Of theses three phases, most of the control
00:08:56.26 and regulation of translation
00:08:59.03 occurs at the initiation phase over here.
00:09:01.01 where ribosomes load onto the message. So given that,
00:09:05.18 I'd like to briefly describe how initiation works,
00:09:07.20 and then we will see how it is regulated in certain cases. So in the eukaryotic cell,
00:09:12.14 initiation is a multi-step process which begins with assembling a cap-binding complex.
00:09:19.11 Eukaryotic mRNAs have on their five prime end a specialized cap structure,
00:09:24.09 which then binds to these cap-binding proteins eIF4E
00:09:29.12 and eIF4G. The presence of these proteins being bound
00:09:33.24 they can also then interact with a protein on the three prime end of the RNA on the poly-A tail,
00:09:38.18 so called poly-A binding protein.
00:09:40.20 That complex then is able, in a subsequent step, to recruit
00:09:45.15 the small ribosomal subunit
00:09:47.29 to the mRNA. And that small ribosomal subunit is loaded onto the mRNA,
00:09:52.07 in what is called a 43S complex. And its called that because it includes other factors,
00:09:57.26 including initiator tRNA, and some translation initiation factors.
00:10:01.19 In a third step, that complex then scans down the mRNA
00:10:07.00 looking for an AUG codon, and typically the first AUG is used in eukaryotic mRNAs,
00:10:12.11 , and once that AUG is recognized, there are various rearrangements of that complex,
00:10:17.16 including GTP hydrolysis, which allows for the 60S, the large subunit of the ribosome to join,
00:10:23.19 and then enter into the elongation phase.
00:10:28.09 So I want to emphasize then these two phases, one assembly of the cap-binding complex,
00:10:32.16 as an initial stage to build an mRNA protein complex,
00:10:36.16 which can receive ribosome, and then a second step
00:10:40.02 where the ribosome is recruited, typically the small subunit first,
00:10:43.21 and then recognition of the AUG.
00:10:49.08 Now in some cases, ribosomes can be loaded on messages independent of the cap structure.
00:10:54.22 And the most extreme version of this is what is called an internal ribosome entry site, or an IRES
00:11:01.00 where ribosomes directly interact
00:11:03.26 with the message. So these are RNA structures, which have been most described in viral RNAs,
00:11:12.11 and they, instead of interacting with, in the most extreme version here,
00:11:17.04 they interact directly with the ribosome. This is what is called an IRES from
00:11:20.23 the cricket-paralysis virus, and this has a specific RNA structure,
00:11:24.02 which then binds directly to the small subunit of the ribosome,
00:11:27.08 recruiting it to the message, and allowing initiation of translation.
00:11:31.08 There are wide varieties of these types of IRESs,
00:11:33.08 for example in Hepatitis C virus, this IRES exists which recruits the ribosome
00:11:39.01 but does so using one of the initiation factors
00:11:41.19 which is present in the normal canonical complex.
00:11:47.08 We should expect that many cellular mRNAs
00:11:49.10 include features like internal ribosome entry sites
00:11:52.14 and one area of ongoing research is describing those in eukaryotic mRNAs.
00:12:00.09 Now one of the properties of translation is that
00:12:02.23 different mRNAs translate to different rates. And that is due to really two kinds of features.
00:12:08.06 One is intrinsic differences in how the mRNA interacts with the translation machinery.
00:12:13.06 For example, if we look at a mRNA
00:12:15.28 there are certain differences which can affect its initiation process.
00:12:20.15 For example, some mRNAs can have strong secondary structure in their five prime end,
00:12:25.01 which prevents the ribosome from loading on, or scanning to the AUG.
00:12:29.15 Other mRNAs can have what are called upstream ORFs.
00:12:33.24 This would be an AUG, and a stop codon, before the normal AUG.
00:12:39.06 So that ribosomes which are loading on the message, actually start at this AUG,
00:12:43.26 stop, fall off, and therefore limit how many ribosomes can get to the normal AUG.
00:12:50.25 IRES elements can exist in RNAs, which increase their translation.
00:12:54.18 And then, the context of the AUG, that is the local sequence context
00:12:59.02 can affect how well the 40S ribosome can recognize this AUG during the scanning process.
00:13:03.28 And all of these features then, can give kind of personalized rates of
00:13:09.00 translation to mRNAs, simply by affecting how that mRNA interacts with
00:13:13.21 translation machinery directly.
00:13:18.19 Layered on top of those intrinsic differences are regulatory components which
00:13:24.11 are targeted to specific mRNAs. So, for example, mRNAs can have proteins
00:13:29.19 which bind to their five prime end,
00:13:31.07 which can actually block the scanning of ribosomes. And therefore block initiation.
00:13:36.09 Similarly, at the three prime end, we can have either
00:13:39.18 RNA binding proteins or micro-RNAs, which are small RNAs that recognizes specific
00:13:45.00 sequences in the RNA, and deliver a so-called RISC complex,
00:13:50.07 or RNA-induced silencing complex to the message, and that these complexes,
00:13:54.05 either protein or micro-RNA derived,
00:13:55.25 can inhibit or enhance the function of various translation initiation factors.
00:14:04.03 Finally, some mRNAs contain within them
00:14:07.14 binding sites for specific proteins, which when bound,
00:14:11.23 actually lead to modifications of the mRNA.
00:14:15.07 And most notably, with regard to translation, are proteins which bind the 3 prime end of mRNAs,
00:14:20.06 and then promote the extension of the 3 prime poly-A tail. Extending the 3-prime poly-A tail
00:14:26.10 can promote translation by a number of mechanisms.
00:14:29.04 But most likely, it loads the poly-A binding protein,
00:14:32.23 which then interacts with the cap complex, providing a better mRNA-protein complex
00:14:39.16 for the loading of ribosomes.
00:14:44.23 Translation can also be regulated in a global way
00:14:48.08 by alterations in the functions of specific initiation factors.
00:14:52.01 So for example, when growth is stimulated,
00:14:56.12 The TOR pathway is enhanced. And that leads to phosphorylation
00:15:00.09 of a protein that binds the cap binding protein.
00:15:03.14 So when that protein is phosphorylated,
00:15:05.14 it lets go of the cap-binding protein.
00:15:08.14 You end up with more cap binding protein and that promotes the loading
00:15:12.17 of initiation factors on the 5' end of mRNAs.
00:15:15.27 So you increase translation generally.
00:15:17.28 Similarly, during a wide variety of stresses,
00:15:21.07 this initiation factor eIF2, which is involved in delivering the initiator tRNA
00:15:27.08 to the ribosome, gets phosphorylated,
00:15:29.26 and when it is phosphorylated, it gets stuck in a GDP-bound form
00:15:34.26 with a translation factor called eIF2b, Which reduces your ability to form these 43s
00:15:40.29 pre-initiation complexes. So there is global control of translation
00:15:45.12 by the phosphorylation or modification various key translation factors.
00:15:53.00 Now one important feature of these global control systems
00:15:55.17 is that they don't necessarily affect every
00:15:58.01 mRNA in the same manner.
00:15:59.10 And I want to illustrate that by using a simple mathematical model
00:16:03.16 for the process of translation initiation. And the larger point I want to make here
00:16:08.08 is that the global control of translation effects mRNAs
00:16:12.21 differently, due to their different rate limiting steps during initiation.
00:16:16.05 So let's consider the process of translation
00:16:19.06 in a very simplified view, of having three discrete steps.
00:16:23.13 In the first step, we would assemble this cap binding complex
00:16:27.11 on the mRNA. In the second step, the small
00:16:31.05 ribosomal RNA, small ribosomal subunit, would scan down the
00:16:34.27 the message, find the AUG codon. And the third step,
00:16:39.10 the large ribosomal subunit would come in and allow for protein production to begin.
00:16:44.10 Now, we can mathematically model this,
00:16:48.05 and in doing so, we are going to assume that this third step,
00:16:51.10 the joining of the 60s subunit is relatively fast
00:16:53.25 compared to the other two.
00:16:54.27 And there is evidence in vivo that suggests that is generally true.
00:16:58.07 Alright, so what happens if we do that, and then we look at the amount of protein production
00:17:04.18 for how those rates change.
00:17:06.09 And that is illustrated in this slide
00:17:08.18 where this is basically a graph showing
00:17:10.22 protein production as a function of the rate of assembly
00:17:14.18 of the translation complex, and
00:17:16.14 and the rate of scanning and AUG recognition.
00:17:20.13 So let's consider two different mRNAs
00:17:22.24 so if we look down here, this axis here,
00:17:26.22 is the rate of assembly, so consider an mRNA
00:17:29.10 whose assembly is slow. So this would be right here.
00:17:34.07 If we change the rate of scanning an AUG recognition from
00:17:37.27 fast to slow, it doesn’t really change the amount of protein production.
00:17:42.12 And that is simply because the rate limiting step
00:17:45.03 is the rate of assembly of the initial translation complex.
00:17:47.25 Conversely, consider an mRNA where the rate of assembly is fast.
00:17:53.02 Ok, so that would be out here.
00:17:54.24 Now, if we change the rate of scanning and AUG recognition from fast to slow, we get a
00:18:01.11 significant change in the amount of protein produced.
00:18:03.16 When the rate of scanning and AUG recognition is fast, there is a lot of protein.
00:18:07.14 Now if you decrease it, there is less protein.
00:18:09.24 Where as for this RNA, there is relatively little change at all.
00:18:13.07 OK. So, the important point here, is because you have different rate limiting steps.
00:18:18.01 changing a given step, may affect an mRNA the same
00:18:23.18 or different from other mRNAs. And I want to emphasize this is a general
00:18:27.06 principle of any pathway which has multiple steps.
00:18:31.02 Where different ones can be rate limiting for different substrates. And this is a reoccurring theme
00:18:37.02 both in translation and also in the steps of mRNA degradation.
00:18:40.17 which we will discuss in a few minutes.
00:18:46.02 Now, some mRNAs are not being translated all the time
00:18:49.15 in eukaryotic cells, and they are actually repressed, perhaps to be translated
00:18:53.05 at a different time or a different place.
00:18:55.00 And that repression of mRNAs commonly involves the formation of
00:18:59.17 what I would call a non-functional mRNP, or mRNA protein complex.
00:19:05.07 Interestingly enough, a common theme in
00:19:07.14 those types of complexes, is that the cap structure is bound by this cap-binding protein
00:19:11.17 eIF4E that we have been discussing. But now instead of being partnered with eIF4g
00:19:16.05 it is partnered with other proteins
00:19:18.05 , for example Cup in drosophila or Maskin in Xenopus,
00:19:23.09 and that those then bind to other RNA-sequence specific binding proteins
00:19:29.24 to create a complex which keeps the cap bound by this cap-binding
00:19:34.07 protein, but is now unable to go on by binding to other translation factors
00:19:39.28 and enter translation. One of the reasons this is probably such a prevalent type of complex
00:19:45.07 for repression, is that by binding the cap here with
00:19:48.11 a cap binding protein, we are actually going to block the major pathway of mRNA degradation.
00:19:53.04 This not only represses the mRNA for translation,
00:19:56.04 but keeps it available by preventing its degradation.
00:19:58.23 And I'll discuss that in a few minutes here when we talk about degradation.
00:20:06.19 Now these repressed mRNAs often
00:20:08.24 end up in large RNA-protein granules in the cytoplasm.
00:20:12.21 So for example, repressed mRNAs often end up in structures in somatic cells,
00:20:18.04 be they yeast, humans, plants, C. elegans, every eukaryote looked at so far
00:20:23.01 which are called P-bodies.
00:20:24.14 And similarly, mRNAs that are repressed in germ cells, where maternal
00:20:30.19 mRNAs are made by the mother, deposited in the oocyte,
00:20:35.07 and used in the embryo in certain places and at certain times,
00:20:38.19 to direct development, are stored in what are called germinal granules.
00:20:42.15 As I will talk about in my second lecture, these different types of RNA granules
00:20:47.26 are all related to each other, and use a common set of RNA-binding
00:20:52.10 proteins to regulate the translation and even degradation of many of these mRNAs.
00:20:56.03 And that how these different granules function, will be the subject of my second lecture.
00:21:05.14 Now following translation, every mRNA
00:21:07.20 eventually gets degraded.
00:21:09.07 And I would like to emphasize three reasons why RNA turnover is actually an interesting area to look at.
00:21:15.10 First, this controls the levels of mRNA and is a common site of regulation.
00:21:21.15 Obviously, if you make RNAs at different rates, you will have different concentrations,
00:21:25.29 But the rate at which they are degraded has tremendous influence on the steady state pool.
00:21:33.01 Second, RNA turnover plays a significant role
00:21:36.01 in defining the transcriptome by degrading many mRNAs that are aberrant.
00:21:41.22 So for example, mRNAs which contain nonsense codons
00:21:46.06 shown here as red, so you don’t translate the entire open reading frame
00:21:50.26 are recognized and rapidly degraded
00:21:53.02 in eukaryotic cells. This is a process called nonsense-mediated decay.
00:21:56.20 And there are other of these types of quality control pathways
00:22:00.18 which recognize and degrade aberrant or non-functional mRNAs.
00:22:04.24 And this is probably important in both preventing the
00:22:08.04 formation of truncated proteins from this type of mRNA.
00:22:10.23 But in also keeping the pool of mRNA in the cytoplasm
00:22:15.12 restricted to those which are functional and which the cell wants to use to produce proteins.
00:22:23.00 Finally, RNA turnover also plays roles
00:22:24.22 in contributing to defense against virus and parasitic elements, such as retro-transposons
00:22:29.26 , these include things like anti-viral miRNAs, RNase L,
00:22:34.29 and even just competition between the general decay
00:22:37.10 pathway and viral function in many cases.
00:22:44.10 Now, work over the last 20 years has defined the ways by which
00:22:47.24 RNAs are degraded in eukaryotic cells.
00:22:49.15 Eukaryotic RNAs when they are born, have this three-prime poly-A tail
00:22:54.10 as well as the cap structure on their five-prime end.
00:22:57.04 In the first step of degradation, this three-prime poly A tail
00:23:01.06 is shortened by a variety of nucleases.
00:23:03.23 The two most important nucleases I'll be mentioning are a complex called the CCR4
00:23:09.13 not complex shown in blue here, and a second complex referred to as
00:23:13.22 PAN2 and PAN3, which seems to play a more minor role.
00:23:17.07 Following de-adenylation, mRNAs can be degraded from the three prime end
00:23:21.00 by a complex called the exosome,
00:23:23.05 which takes advantage of various cofactors to degrade mRNAs. Or, what is a more common
00:23:29.12 degradation pathway, mRNAs are then decapped
00:23:32.19 where the cap structure is removed by a decapping enzyme
00:23:35.28 followed by five-prime to three-prime degradation of the RNA.
00:23:41.11 Now, one of the unresolved issues
00:23:43.10 is the distribution of mRNAs between these two general RNA decay pathways.
00:23:48.09 In yeast cells where more of these experiments have been done, it is clear that this decapping
00:23:54.03 and the five-prime to three-prime decay pathway
00:23:56.12 is the prevalent pathway by which RNAs are degraded. But we really don’t understand in more
00:24:00.17 complex eukaryote cells, particularly like mammals, which of these decay pathways predominates
00:24:06.11 and whether that is influenced by different cell types
00:24:08.23 or different signaling pathways, or even the diversity of different mRNAs.
00:24:12.20 And this is an area of ongoing research currently.
00:24:17.09 So there are two general pathways of RNA degradation,
00:24:19.23 deadenylation leading to three-prime decay,
00:24:22.03 or decapping and five-prime decay.
00:24:23.22 There are also specialized pathways by which mRNAs are degraded.
00:24:27.02 So again, another example of quality control,
00:24:30.25 if mRNAs don't have a stop codon, they are recognized and degraded extremely rapidly
00:24:35.24 from the three prime end without any deadenylation. This is a process called non-stop decay.
00:24:42.29 Similarly, RNAs which are recognized as having nonsense codons
00:24:46.03 at least in simple eukaryotic cells,
00:24:48.11 are subject to very rapid decapping.
00:24:50.14 so bypassing this normal step of deadenylation.
00:24:53.27 And a third, and growing class of RNA degradation
00:24:58.07 events which are specialized, are endonuclease cleavage events.
00:25:01.12 Which tend to be targeted to specific subclasses of mRNAs
00:25:04.20 that have specific sequences
00:25:06.09 or elements which are recognized and they trigger endonuclease cleavage.
00:25:11.00 Two examples of this I want to highlight,
00:25:12.21 one is cases where miRNAs
00:25:15.21 can trigger cleavage of mRNAs, and this has been described
00:25:19.09 now in a variety of different eukaryotic cells;
00:25:21.16 most prevalently in plants, but also observed in mammalian cells as well.
00:25:27.23 Another class of endonuclease cleavage is yet another of these so-called quality control
00:25:33.10 systems, where when ribosomes are translating down a message,
00:25:36.23 if they get stuck for any reason,
00:25:38.24 so they are stuck in elongation,
00:25:40.16 that triggers cleavage of the mRNA, to rescue that dead-end complex
00:25:44.16 and degradation of the two pieces.
00:25:49.05 So an unresolved area here is
00:25:50.25 how diverse are these types of endonuclease cleavage in eukaryotic cells
00:25:54.25 and how common are these distinct classes.
00:26:01.09 Now mRNA rates can vary substantially between
00:26:04.11 individual mRNAs, and this is just an example of this,
00:26:06.29 looking at three different yeast mRNAs.
00:26:09.05 Here at the zero time,
00:26:11.06 you block transcription of these mRNAs and you can see that some like the PGK
00:26:15.03 message lasts a long time.
00:26:16.15 Others, like the MFA2 message, are
00:26:18.24 extremely unstable and degrade quickly.
00:26:20.21 In mammalian cells, the average rate of decay
00:26:24.04 of mRNAs is about 8 hours, but again those can vary tremendously.
00:26:28.10 For example, the cFOS mRNA has a half-life of about 15 minutes, and is degraded very quickly.
00:26:33.17 Whereas the Beta-globin mRNA, which last for about 17 hours (of a half-life).
00:26:39.04 So there are tremendous differences in these decay rates between individual messages.
00:26:45.00 One feature of mRNAs that controls their decay rates.
00:26:47.16 are what people have referred to as so-called stability elements.
00:26:50.23 These are short sequence elements within mRNAs
00:26:53.04 that control the rates of RNA turnover. So for example, within the c-Fos mRNA
00:26:57.20 there is an AU-Rich element, or so-called ARE, which can be transferred to
00:27:04.01 the beta-globin mRNA and then cause a rapid deadenylation,
00:27:07.13 and rapid decay after deadenylation.
00:27:09.27 And this contrast to the globin message which is normally slow for those two steps.
00:27:15.13 So these so-called stability elements then, are part of the features of mRNAs
00:27:20.00 which dictate their different rates of RNA turnover.
00:27:24.18 Many of these stability elements serve as binding sites
00:27:27.08 for proteins or microRNAs, that then control RNA-degradation.
00:27:31.01 In this example I'll use here today, the PUF3 protein is a protein which binds
00:27:36.19 to a short-sequence element at the three-prime ends of yeast mRNAs.
00:27:40.13 And when it does so, it recruits the CCR4 deadenylase complex,
00:27:44.26 and subsequently can also increase the rate of decapping of that message.
00:27:50.23 And that is a general principle that we see for many of these
00:27:53.00 instability elements, that they recruit these trans-acting factors that then directly or indirectly
00:27:58.12 promote the degradation of a message.
00:28:03.07 Now, one of the striking features of mRNA decay
00:28:05.19 and translation, is how they are intertwined with each other. And I want to
00:28:10.02 highlight this by pointing out
00:28:11.03 the distinct roles of the cap
00:28:12.23 in both translation and RNA degradation.
00:28:16.04 In translation, the cap and the poly-A tail serve as binding sites
00:28:20.27 for RNA-binding proteins which then assemble a complex, which promotes ribosome loading.
00:28:25.21 Conversely, during degradation, these are the two structures,
00:28:30.24 the main nucleases in the cell, target. The poly-A tail is removed
00:28:34.17 by the CCR4 or PAN2 complex, ultimately leading to decapping
00:28:39.19 to the message by the decapping enzyme.
00:28:41.04 Now once the message is decapped, as far as we know,
00:28:44.22 the five-prime and three-prime exonuclease degrades the message extremely rapidly.
00:28:53.10 So in general then, because of these two roles,
00:28:55.11 translation and mRNA decapping, are inversely related.
00:28:59.02 So during translation you have a complex which
00:29:02.04 promotes ribosome loading.
00:29:03.03 During degradation, you lose that complex and you promote degradation.
00:29:07.26 And consistent with that, various observations support
00:29:12.09 this kind of inverse relationship.
00:29:14.10 If you alter the rate of translation initiation, by mutations
00:29:17.08 or alterations to the RNA, you increase the rate of decapping.
00:29:21.19 Similarly, many sequence elements which people have identified as either regulating translation,
00:29:27.26 or regulating decay, do both.
00:29:30.21 For example, the AREs I've talked about, which promote mRNA degradation,
00:29:35.01 often under slightly different conditions, will also repress translation.
00:29:39.18 The way we have interpreted this
00:29:42.14 in general, is that there are really two different types
00:29:46.12 of RNA-protein complexes existing in the cell.
00:29:49.14 And what I will call the translation mRNP
00:29:52.02 and the decapping mRNP, really are distinct.
00:29:55.09 And for example, here, one type, the translation mRNP,
00:30:00.05 and it would have to disassemble this translation initiation complex
00:30:03.16 to form an alternative complex,
00:30:05.19 in this case with the decapping complex in order to be degraded.
00:30:09.03 And the kind of evidence that supports this model
00:30:12.22 comes from either co-immunoprecipitation
00:30:16.07 experiments where RNAs that are bound translation initiation factors are not bound by decapping factors and vice versa.
00:30:22.06 And also, cell biological experiments, which indicate that the mRNA decapping factors
00:30:28.17 are not associated with polysomes, polysomes are the ribosome-mRNA complexes
00:30:33.13 where proteins are being produced,
00:30:34.25 but instead accumulate in these discrete cytoplasmic foci referred to as P-bodies.
00:30:41.26 And these P-bodies, as I mentioned earlier
00:30:43.23 are found throughout eukaryotic cells, this is just an example in mammalian cells
00:30:47.15 and they tend to contain a core set of proteins, including the decapping enzyme,
00:30:52.20 various proteins involved in degradation of those, activation of decapping,
00:30:58.08 as well as the exonuclease, which degrades following decapping Xrn1.
00:31:04.26 In metazoans, these P-bodies can also interact with the microRNA silencing pathway
00:31:11.07 such as argonauts, and GW182
00:31:13.22 and I'll discuss a little bit in my second talk
00:31:16.17 the relationship between these microRNA silencing complexes
00:31:19.23 and these kind of core decapping complexes seen in P-bodies normally.
00:31:30.19 That inverse relationship between translation and degradation
00:31:33.14 has suggested a competition between translation factors
00:31:37.24 and translation repression factors, or P-body mRNPs.
00:31:41.27 And that the function of the mRNA in the cell really then reflects, how the
00:31:46.18 mRNA is partitioned between translation and repression.
00:31:50.15 And I want to emphasize this because
00:31:52.21 in the past we've tended to think about whether an RNA can translate or not
00:31:56.11 really as a function of how well it interacted with initiation factors.
00:31:59.09 But what we have learned now is in fact
00:32:01.15 there is a competing, so called repression complex,
00:32:04.29 which then can also drive the RNA into a repressed state, which then can assemble into
00:32:10.16 into these larger aggregates that we can then see in the microscope.
00:32:13.25 And the function of an mRNA then is really in some sense
00:32:18.14 perhaps dictated by its distribution between these two different competing assembly pathways.
00:32:23.08 So a key point that led to this kind of model
00:32:27.03 has come from an examination of these proteins that are involved in triggering mRNA
00:32:32.01 decapping and P-body formation as studied in yeast. And what we know
00:32:36.24 about those proteins, is that orthologs in other cells
00:32:40.01 are required for the translation repression of specific mRNAs.
00:32:43.21 So for example, a protein called Dhh1,
00:32:46.27 which is involved in promoting decapping in yeast,
00:32:49.17 we know that in many maternal mRNAs
00:32:52.27 is required for the storage of those mRNAs,
00:32:55.08 their translation repression and storage.
00:32:57.03 And we also know that in neurons, where many mRNAs are stored
00:33:00.17 some of those mRNAs, their storage, and preventing them from entering translation
00:33:05.29 requires this Dhh1 protein ortholog.
00:33:10.00 So this repression decay complex is not just
00:33:15.06 for driving RNAs into decapping, it is also for maintaining some mRNAs in a
00:33:19.27 translationally-repressed state.
00:33:23.08 And consistent with that, we know that some of these activators
00:33:26.08 can actually be purified now, and be shown to directly repress translation in cell free extracts,
00:33:32.05 all by themselves, and some cases bind to and inhibit directly
00:33:35.17 the action of some these translation initiation factors.
00:33:42.00 So the bigger point then, is that of course,
00:33:43.28 that these translation repression complexes
00:33:46.29 play a role in translation repression of mRNAs
00:33:48.21 as well as targeting them for degradation.
00:33:52.04 Now, I've already told you that localization of mRNAs
00:33:55.02 requires them to be translationally repressed.
00:33:58.05 So now we've described a complex which is involved
00:34:01.03 generally in translation repression. Might it also have a role in RNA localization?
00:34:05.19 And in general, we don't really know the answer to that question
00:34:08.11 , but there are hints that it will.
00:34:09.28 And I want to highlight that by showing this experiment done in Drosophila embryos
00:34:14.16 where Dcp1, which is a subunit of the decapping enzyme, is actually required for the Oskar mRNA
00:34:20.23 to be localized to the posterior pole of Drosophila embryos.
00:34:24.11 So this is the Drosophila embryo here,
00:34:26.18 growing, and you can see this green here is the Oskar mRNA
00:34:30.10 that is localized to this region of the cell
00:34:31.25 Now if we mutate this component of this decapping enzyme,
00:34:35.00 you can see that the mRNA no longer localizes to the proper region.
00:34:38.28 So this suggests that these components of this translation
00:34:42.22 repression complex, not only can affect decapping,
00:34:45.25 as well as translational control, but at least in some cases then, they might even be involved
00:34:51.06 in repressing the translation of mRNAs, so they can be properly localized.
00:34:58.00 So to step back so far, what I've told you about really
00:35:00.14 are three key areas of cytoplasmic mRNA function;
00:35:03.14 localization of the mRNA, translation, and degradation.
00:35:07.28 And these three processes are coupled,
00:35:10.14 with the general rules that you don't translate until you are localized.
00:35:14.16 And you stop translation before you degrade.
00:35:16.28 They may be coupled even more mechanistically
00:35:20.04 because a similar group of proteins, which are involved in translation repression
00:35:23.25 and decapping, is involved in coupling these processes of translation and degradation.
00:35:30.29 So then, an important question to try to understand how the fates of different mRNAs
00:35:35.24 are controlled, to give different types of localization
00:35:40.00 different types of translation, and different rates of degradation
00:35:43.08 and at the simplest level, we can think about this, is really,
00:35:49.00 how the mRNA interacts with these different machines in the cell.
00:35:52.00 That is the localization is going to be dictated by how the mRNA
00:35:55.15 and the proteins associated with it, interact with motors
00:35:58.12 and anchors in the cell. How well the mRNA translates is going to be influenced by how well
00:36:03.14 it interacts with translation factors, either directly or indirectly.
00:36:06.28 And how well it degrades
00:36:08.20 is going to be influenced by its interaction with the degradation machinery.
00:36:11.28 So an important principle to understand then is
00:36:15.07 how do mRNAs interact with these different machines
00:36:17.21 and how is that different between different messages.
00:36:22.17 First, to highlight something we've already talked about,
00:36:25.02 there are intrinsic differences between different mRNAs.
00:36:28.00 So we have already talked about how, with translation,
00:36:31.27 some mRNAs can have IRESs, which allows them to interact directly with the translation machinery,
00:36:37.03 bypassing the need for certain translation initiation factors.
00:36:40.29 Similarly, there are examples where RNAs act directly with the RNA-decay machinery,
00:36:46.24 and therefore influence their fate.
00:36:48.06 For example, in mammalian cells, the Rrp41 mRNA, has a high affinity binding site
00:36:55.19 for the DCP2 protein, this is the decapping enzyme.
00:36:58.05 And so the presence of that sequence leads to this mRNA being rapidly degraded.
00:37:04.07 So these are intrinsic differences with how they interact
00:37:06.05 with the cytoplasmic machinery.
00:37:09.21 Then we also have layered on top of that again
00:37:11.15 sequence-specific RNA-binding proteins.
00:37:13.26 And how they interact with these machinery as well.
00:37:16.29 So I just want to give two examples of this.
00:37:19.07 So in mammalian cells, there is a protein called HuD, which is a RNA binding protein,
00:37:24.16 that binds to these ARE elements again, and once it does,
00:37:27.28 it also has a region, which can bind to one of the initiation factors
00:37:32.17 in the cap-binding complex, and therefore promote faster rates of translation initiation.
00:37:38.00 So an RNA-specific binding protein binds to the message,
00:37:43.20 recruiting the translation machinery.
00:37:45.19 You see the exact same process with mRNA degradation,
00:37:48.16 here is a RNA-binding protein MPT5,
00:37:52.04 which binds to certain mRNAs in yeast cells,
00:37:54.20 and then has direct interactions with components of this CCR4 de-adenylase complex.
00:38:00.13 So this protein recruits the nuclease to the message
00:38:03.18 and leads to much faster rates of deadenylation.
00:38:07.20 So direct interactions between RNA-binding proteins
00:38:09.28 and either translation machinery, or mRNA degradation machinery.
00:38:15.08 And we might ask, how much of these kinds of regulation should we expect?
00:38:21.04 And we should expect a lot of it.
00:38:22.24 If we look in the genomes of organisms,
00:38:25.17 there is a tremendous number of RNA-binding proteins.
00:38:28.02 In the initial drafts of the human genome
00:38:30.04 predicted greater than 1,000 RNA binding proteins, and this is probably
00:38:33.24 and underestimate, because we know that many proteins bind RNA,
00:38:37.16 which don't have sequence motifs, which allow us to identify them as RNA binding proteins.
00:38:41.28 So the yeast genome, has greater than 600 RNA binding proteins.
00:38:50.10 In general, these RNA binding proteins are probably going to bind mostly to the 3-prime UTR;
00:38:54.21 the three prime untranslated region.
00:38:56.20 And that is simply for two reasons: One is, if you think about the
00:39:02.16 the five prime end of the message, or the open reading frame, these are
00:39:06.22 constrained evolutionarily, by their function in translation.
00:39:09.20 The ORF has to maintain the proper coding region for the
00:39:12.11 protein, and, in both cases,
00:39:15.03 the ribosome has to be able to pass through these regions.
00:39:17.15 Therefore any regulatory protein which binds, will be dislodged,
00:39:22.10 by the passage of ribosomes.
00:39:23.21 So this puts constraints on the ability of these regions to function as regulatory
00:39:28.13 sites for RNA binding factors.
00:39:29.29 In contrast, the 3-prime UTR, really is free to evolve new structures, new
00:39:34.12 binding sites, and factors that bind here are not going to be displaced
00:39:38.05 by transversing ribosomes, and can maintain
00:39:41.19 their association with the message, and therefore regulate its function
00:39:44.17 in a more efficient manner.
00:39:48.00 So how many factors should we expect to bind, there?
00:39:50.17 And we don't really know the answer to this question.
00:39:55.17 The average, using humans as an example,
00:39:57.13 the average 3-prime UTR, is about 740 nucleotides,
00:40:01.03 although it can vary tremendously from 60 to 4,000.
00:40:06.10 So how many factors might you expect to bind here?
00:40:08.27 Well, a low estimate might be one factor for every 100 bases.
00:40:13.24 But we know that, in general, proteins bind to
00:40:17.28 sequence elements around 10, and microRNAs bind to around 20
00:40:23.07 nucleotides, so maybe a high estimate would be 1 factor binding for every 20 bases,
00:40:28.06 which will lead to 37 factors bound.
00:40:30.22 And this is probably unrealistic.
00:40:32.09 But even at the low estimate, of 7 proteins or factors being bound to the 3-prime UTR,
00:40:40.05 you can see that the average message
00:40:42.02 will have multiple regulatory factors bound to its 3-prime end.
00:40:45.07 And therefore its control of localization, translation, and degradation,
00:40:50.07 will probably be a summation of the impacts of all these different factors.
00:40:53.24 And not just the impact of a single, sole protein.
00:41:02.00 Now, a few other principles of these RNA-binding proteins
00:41:05.06 which are important to consider: First they can affect more than one process.
00:41:08.19 I want to go back to the Puf3 protein in yeast that we use as an example of factors that binds
00:41:14.07 and promotes RNA-degradation.
00:41:15.20 But we also know that this protein also binds and targets a subset of RNAs to
00:41:21.04 to localize to the surface of the mitochondria.
00:41:23.01 So this single protein then
00:41:24.29 can both promote degradation and target RNAs
00:41:28.10 for proper subcellular localization.
00:41:30.13 And there are many examples of this,
00:41:32.22 where RNA proteins can affect more than one process in the cytoplasm.
00:41:40.11 Proteins associated with RNAs range from
00:41:43.18 very general to highly specific.
00:41:46.05 This is a diagram of the number of mRNAs
00:41:48.28 associated with about 40 different RNA-binding proteins
00:41:53.06 based on co-immunoprecipation from yeast cells.
00:41:56.24 And what you can see is that some proteins,
00:41:59.11 for example out here the poly-A binding protein,
00:42:01.23 which we think of as relatively general, binds to approximately 1,000 mRNAs.
00:42:06.21 And down here at the other extreme, some proteins which we think would bind
00:42:11.07 to RNAs only bind to a few, less than 5.
00:42:16.26 So, RNA binding proteins can be either highly general,
00:42:20.20 binding to many, many factors in the cell or highly specific.
00:42:23.18 And one thing we don't understand well at all
00:42:25.15 is really, what are the diversity of different RNP types,
00:42:28.15 and how does that affect the function of mRNAs in the cell.
00:42:34.14 But one thing we do know, is that in many cases,
00:42:37.11 RNA-binding proteins then co-regulate mRNAs, which have related function.
00:42:41.17 So for example, so using the Puf3 protein as an example again, the Puf3
00:42:46.22 protein binds to about 200 mRNAs in yeast cells. And essentially all 200 of those
00:42:51.06 proteins encode proteins involved in mitochondrial function.
00:42:54.01 So it makes sense that they would actually A) be coordinately controlled for their degradation.
00:43:00.08 And, for their targeting for specific subcellular localizations.
00:43:03.20 And consistent with that, Puf3 targets these mRNAs to the mitochondrial surface,
00:43:08.12 and promotes their degradation under conditions
00:43:12.02 where mitochondria is important in the physiology of of yeast cells.
00:43:20.17 So in the last few minutes, what I want to do is talk about mRNP assembly;
00:43:24.06 the different stages and how dynamic it is. And this is important because, I think,
00:43:29.21 that what I will try to convince you of
00:43:31.11 in the last few minutes, is that the proteins associated with mRNAs,
00:43:35.16 are really important in dictating
00:43:37.13 how well it is translated, degraded,
00:43:38.28 and where it is localized within the cell.
00:43:41.10 And so we need to think then about what is the process by which these proteins are assembled
00:43:45.28 on mRNAs, and how does it change.
00:43:50.14 And there are a few general principles I want to highlight here.
00:43:53.20 First, that mRNP assembly begins in the nucleus.
00:43:56.20 So I began the talk by mentioning this fact,
00:44:00.23 but that many mRNA-binding proteins are imported into the nucleus,
00:44:04.16 and assembled with the nascent transcript,
00:44:06.11 and then are exported with the RNA out into the cytoplasm.
00:44:09.16 And in fact, as we study more and more
00:44:12.12 proteins that bind to mRNA, this is becoming more of a common theme.
00:44:16.22 Where many, many factors are loading on the message in the nucleus
00:44:19.26 probably as the RNA is being produced co-transcriptionally.
00:44:27.00 Once the mRNA is exported to the cytoplasm,
00:44:29.27 simply being in a different subcellular compartment can promote some transitions in the
00:44:34.24 RNA-protein complexes.
00:44:37.02 For example, in the nucleus, when there is a certain complex that binds to
00:44:42.20 the five-prime end, called the nuclear-cap binding complex,
00:44:45.04 and once the mRNA reaches the cytoplasm
00:44:48.23 the presence of RAN-GDP, which is a protein-GDP complex
00:44:54.06 which is at high levels in they cytoplasm,
00:44:56.21 whereas in the nucleus you tend to have a related RAN-GTP complex.
00:45:00.21 The presence of that RAN-GDP causes loss of that nuclear-cap binding complex
00:45:05.05 and loading of the cytoplasmic cap binding complex we talked about
00:45:08.28 the eIF4E and 4G
00:45:10.27 which promotes translation onto the messages.
00:45:13.02 And that simply occurs because the mRNA is now in a compartment
00:45:17.00 with a different concentration of RAN-GDP.
00:45:19.11 Similarly, localization to specific subcellular regions can affect the exchange of proteins.
00:45:25.27 For example, the beta-actin mRNA that I mentioned in the beginning, which is targeted
00:45:31.27 to the leading edge of a crawling fibroblast,
00:45:36.24 when it reaches out there, a protein which targets it there
00:45:40.07 called the ZIP-code binding protein, is phosphorylated by the Src kinase,
00:45:45.12 which is localized to that part of the cell, and is caused to be released from the mRNA.
00:45:49.25 So simply being...
00:45:51.01 because different compartments of the cell have different biochemical properties,
00:45:54.22 that can change the proteins which are associated with the message
00:45:57.20 at different places within the cell.
00:46:03.06 Another way that proteins are exchanged is through the process of translation.
00:46:07.01 When the message comes out of the nucleus,
00:46:09.07 presumably there are many proteins that bind to the coding region.
00:46:12.01 And one example of that would be this exon-junction complex,
00:46:15.07 which is deposited at splice junctions, which are predominately found within the coding region.
00:46:20.01 But when ribosomes reach an exon junction complex, they disassociate it
00:46:25.21 through direct interactions. And they cause that to be bumped off the message.
00:46:30.04 And so the entry of mRNAs into translation, and the movement of ribosomes
00:46:34.21 down the message, cause the loss of essentially the proteins
00:46:38.00 which are bound to the coding region from the mRNA complex.
00:46:43.24 Whereas, mRNAs which are bound to the three-prime UTR,
00:46:46.09 we anticipate, remain bound relatively stably.
00:46:49.19 Although there is no direct measurements
00:46:51.04 for what the dynamics of those proteins are within cells.
00:46:58.08 Finally, mRNAs can be dynamic in ways that we really don't understand yet,
00:47:03.01 that involve large-scale rearrangements in the set of
00:47:05.12 proteins associated with it. One thing
00:47:07.09 I will talk about in my second lecture is that during times of stress
00:47:10.24 mRNAs can exit translation, and assemble a different complex of proteins
00:47:15.00 involving the translation repression complex
00:47:17.07 and decapping factors we talked about.
00:47:19.19 And when they do that,
00:47:21.24 they actually lose all these translation factors, which are currently on the message.
00:47:25.06 These mRNAs that are repressed then can re-enter translation at later times,
00:47:29.21 then, losing all these factors, and reassembling a new translation complex.
00:47:34.16 So there can be large-scale rearrangements of the mRNA-associated proteins.
00:47:38.23 The mechanism by which these occur, and their frequency,
00:47:41.25 really are not well understood at all yet.
00:47:44.03 But we know that they can occur.
00:47:48.17 Finally, the proteins associated with mRNAs
00:47:50.19 are dynamic, in that they are modulated by modifications.
00:47:54.11 For example, this one I want to give here, a protein called BRF1, which
00:47:59.16 binds the 3-prime end of mammalian messages
00:48:02.06 and promotes their degradation by the de-adenylase, can be phosphorylated in response
00:48:07.16 to various signal transduction pathways, and once phosphorylated,
00:48:11.11 that recruits a protein that binds to that phospho-protein complex,
00:48:16.05 blocking the ability of BRF1 to recruit the de-adenylase.
00:48:21.03 Now, so not only are the proteins dynamic then, they can be modulated by modification.
00:48:26.27 And the list of modifications of RNA-binding proteins
00:48:29.27 has grown extremely long, and continues to grow.
00:48:33.10 We now know of many proteins that are methylated,
00:48:35.23 acetylated, ubiquitinated, can be O-GlcNac modified,
00:48:40.27 they can be ADP-ribosylated, sumoylated, and in an example I already gave you, phosphorylated.
00:48:47.08 So what this means then, is
00:48:48.11 that even for mRNA-binding proteins which remain stably bound to the message,
00:48:52.20 their function can be modified dramatically in response to various
00:48:56.11 signal transduction pathways or environmental cues,
00:48:59.04 simply by changing their modification status.
00:49:01.09 Finally, the mRNA itself is modified.
00:49:07.19 As we talked about during the process of degradation, mRNAs which are born have
00:49:12.16 long poly-A tails, and then lose them.
00:49:15.04 This is really a modification of the RNA sequence,
00:49:17.09 But then also affects the RNP composition
00:49:20.21 because it removes binding sites for the poly-A binding protein.
00:49:23.26 Other modifications can happen to this RNA.
00:49:27.08 Poly-A tails can be added back, which provides new sites for poly-A binding protein,
00:49:32.02 and therefore can nucleate the assembly of new translation complexes.
00:49:35.00 In the last two years we have also learned that mRNAs can undergo a process called uridinylation,
00:49:40.23 where poly-U tails are added to the 3-prime end of the message.
00:49:44.12 And this can recruit at least some
00:49:47.00 components of the RNA-degradation pathway,
00:49:48.27 but, because this is a relatively new discovery,
00:49:51.29 what the roles of poly-uridinylation are, and the diversity of factors that bind to those,
00:49:57.01 really remain to be discovered.
00:49:59.07 Finally, we have to anticipate that there are other modifications to RNAs,
00:50:03.05 perhaps methylation of nucleotides that have yet to be discovered.
00:50:08.17 But, by changing the covalent structure of the RNA itself,
00:50:12.08 that can also influence the proteins that are bound,
00:50:15.04 and its interaction with these various machines regulating translation,
00:50:18.03 degradation, and localization.
00:50:23.01 Ok. So, I've tried to give you a big picture then today,
00:50:27.07 of the three important processes in the cytoplasm of eukaryotic cells.
00:50:30.29 mRNA localization, translation, degradation. And these are really very important,
00:50:36.02 for regulating the amount, the duration, and the location of protein production
00:50:40.14 in complex eukaryotic cells.
00:50:43.03 One principle that has emerged from studying these, is that they are actually all interconnected.
00:50:48.07 With localization requiring the message to be translationally repressed until it is localized.
00:50:52.15 Translation being related to degradation, because you stop translation before you degrade it.
00:50:58.03 And then the proteins that are involved in regulation of translation,
00:51:02.08 can play multiple roles in repressing translation until you are localized,
00:51:07.21 stopping translation for degradation,
00:51:09.15 or repressing translation for mRNA storage
00:51:12.08 for cases where mRNAs need to be stored, and translated at later times.
00:51:17.17 A third important principle really is that, the proteins that are associated with the message,
00:51:21.22 as well as the message's interaction with
00:51:24.03 the translation and degradation machinery directly,
00:51:27.01 really are what define the mRNA's specific properties
00:51:29.18 of localization, translation rate, and half life.
00:51:33.09 And that an important goal would be to understand
00:51:36.12 what these proteins are, and how they function.
00:51:41.17 And finally, these RNPs are dynamic,
00:51:44.13 and are modulated by many types of transitions, both in
00:51:48.03 composition, and modification, as well as in modification of the RNA itself.
00:51:52.05 And so that we can't think of the RNA-protein complex as a static structure.
00:51:55.26 We have to think of it as one that is constantly under flux,
00:51:58.13 at many different levels of control.
00:52:04.00 So this leads to a number of different issues in the future here.
00:52:08.10 You know, one of the things that we don't understand very well is
00:52:10.18 what the diversity and dynamics of mRNP types are, and how
00:52:13.21 that relates to RNA function.
00:52:15.04 You know, in many ways we can think about RNA-protein complexes as
00:52:19.00 analogous to DNA-protein complexes.
00:52:21.16 But the difference is that DNA is relatively homogeneous.
00:52:25.01 Whereas RNAs can be extremely heterogeneous, they can fold in much
00:52:29.11 more complex and diverse structures, and so it could be there is a tremendous
00:52:33.01 diversity of different types of RNP types, and we just don't understand those yet.
00:52:40.13 We don't understand these modifications.
00:52:43.01 Again, analogous to DNA, you know, there is an idea of a histone code, where different
00:52:47.24 modifications to chromatin, influence whether the DNA is available,
00:52:53.05 or inaccessible for transcription.
00:52:55.08 Is there a corresponding code for mRNAs?
00:52:58.17 Where different types of modifications on these mRNA binding proteins,
00:53:02.01 drive RNAs either into repressed states,
00:53:04.27 or translation states.
00:53:06.10 And that therefore, regulation of those modifications become
00:53:09.13 important control points for regulating
00:53:11.23 either the general levels of translation, or the translation of specific mRNAs within the cell.
00:53:16.27 And finally, one has to hope that,
00:53:18.27 as we continue on in this field,
00:53:20.27 by developing an understanding of these principles, we might be able to
00:53:25.17 develop a predictive model of mRNA function, based on sequence, which integrates
00:53:29.02 all these different properties, and would allow us to be able to predict
00:53:31.27 where mRNAs will end up in the cell,
00:53:34.26 how frequently they will be translated, and for how long they will last on average.
00:53:40.24 And with that, I thank you for your attention, and I'm done.
00:53:44.10

Part 2: P-bodies and the mRNA Cycle

00:00:00.00 Hello. My name is Roy Parker. I am Professor at the University of Arizona,
00:00:04.08 and an investigator at the Howard Hughes Medical Institute.
00:00:06.26 In my two talks today, I'll be talking about the life of the eukaryotic mRNA.
00:00:10.29 In my first talk, I discussed the mechanisms by which mRNAs are translated,
00:00:15.11 localized, and degraded in eukaryotic cells. In this talk, I'll be discussing P-bodies,
00:00:22.01 and what we call the mRNA cycle.
00:00:25.01 And what that tells us about the regulation of eukaryotic mRNAs.
00:00:31.23 This slide here shows a human liver cell in culture,
00:00:37.26 and you can see these bright regions in the cytoplasm, and these red dots.
00:00:42.03 And these are what we call P-bodies, or cytoplasmic processing bodies.
00:00:46.23 And what we have learned from studying these processing bodies,
00:00:50.11 is that they tell us about a dynamic cycle of mRNAs in the cytoplasm,
00:00:54.15 which affects their function.
00:00:55.27 And that cycle is cartooned in this slide, and has the following key properties.
00:01:01.14 First, mRNAs can exist in a translating state, where they are associated with ribosomes,
00:01:06.05 making proteins.
00:01:07.05 But under certain conditions they can exit that state,
00:01:09.29 lose their translation factors and ribosomes,
00:01:13.04 and assemble what we call a P-body mRNP,
00:01:15.20 which is a translationally repressed mRNP,
00:01:17.29 Which can aggregate in these larger P-bodies.
00:01:20.28 mRNAs within P-bodies can either be destroyed,
00:01:25.00 or, they can return to translation by assembly of a new translation initiation complex,
00:01:30.04 and then recruitment of a ribosome.
00:01:33.02 Now, there are three features of this cycle in P-bodies
00:01:37.11 that I find particularly interesting, and worthy of study.
00:01:41.10 First, the transitions between these different states
00:01:44.19 in the cell are highly likely to play important roles in the regulation of translation
00:01:49.17 and degradation, and perhaps even localization of RNAs in the cell.
00:01:53.16 Obviously, if mRNAs are associated with ribosomes and translation factors
00:01:57.15 they can make proteins. But when they are assembled in translation repression complexes
00:02:01.23 and associated with RNA-degradative complexes,
00:02:05.02 they are more likely to be targeted for destruction.
00:02:07.10 Moreover, we also think that this might play some role in localization,
00:02:11.09 because these components in these P-body structures,
00:02:14.06 actually show similarity to RNA-transport granules in a variety of different cases.
00:02:20.24 So this cycle, probably plays a significant role in just the regulation
00:02:24.21 of translation, degradation, and localization of RNAs in cells.
00:02:29.04 A second interesting feature of this cycle,
00:02:33.01 is that P-bodies, a key component of this cycle,
00:02:37.07 show overlap with other RNA-protein granules that are very important in biology.
00:02:42.08 For example, maternal RNA-granules, or germinal granules,
00:02:47.00 are complexes of maternal mRNAs and proteins found in the oocytes or the embryos,
00:02:53.12 of a wide variety of species. And those maternal mRNAs
00:02:56.11 are made by the mother and then during the development of the embryo,
00:02:59.28 are translated in specific places and at specific times,
00:03:03.21 in order to traject the development of that early embryo.
00:03:07.04 And so the storage, and subsequent translation,
00:03:09.18 at the right time and place is extremely important.
00:03:11.16 And those granules share many components with P-bodies,
00:03:16.03 which are found in every somatic cell that has been examined so far,
00:03:19.07 from yeast to humans.
00:03:22.00 Similarly, in neuronal granules,
00:03:23.19 which play an important role in synaptic plasticity,
00:03:25.29 and transport RNAs out to various synapses, also share components
00:03:32.02 with P-bodies. And also share components with germinal granules.
00:03:35.20 So all three of these types of RNA-granules are related,
00:03:38.21 and probably have an underlying, similar, biochemical and compositional function.
00:03:46.17 Finally, the third reason that I find these interesting,
00:03:49.07 are connections between viruses and the mRNA cycle.
00:03:52.01 So in several cases, components of P-bodies,
00:03:55.19 or stress granules, another granule in this cycle we will talk about,
00:03:58.21 are required for viral life cycles.
00:04:00.21 For example, components of P-bodies are required for
00:04:04.29 retrotransposons, which are retroviral-like elements in yeast.
00:04:09.25 And components of stress granules,
00:04:11.20 such as the protein called DDX-3, is required for translation of HIV
00:04:16.13 genomic RNAs, as well as for hepatitis C viral life cycles.
00:04:22.00 And we think these genetic roles are actually important,
00:04:26.08 because in some cases the viral complexes, or host anti-viral factors
00:04:32.19 accumulate in these structures. So for example, what we are looking at here,
00:04:36.11 these are yeast cells, and the green are actually markers of P-bodies,
00:04:40.25 and the red here, are actually showing newly assembled viral particles
00:04:46.17 for this retrotransposon Ty3. And you can see, while they don't overlap completely,
00:04:52.08 these viral particles tend to be found in conjunction with partial overlap with P-bodies.
00:04:59.20 So my lab has then been interested in trying to understand
00:05:02.20 what the properties and functions of P-bodies are,
00:05:05.29 and how it relates to these dynamic transitions that RNAs can undergo,
00:05:09.16 in regulating their translation and degradation.
00:05:15.15 Now, work from a wide variety of labs has identified many of the components
00:05:21.05 in P-bodies, although we still do not understand the entire complex
00:05:24.14 that is present in these particles.
00:05:26.28 So from yeast to mammals, there is a core set of proteins,
00:05:30.27 which include decapping enzymes,
00:05:33.03 as I talked about in my other talk, plays a role in degrading RNAs
00:05:37.10 by decapping. As well as various proteins which either can repress translation,
00:05:41.08 or promote decapping, as well as an exonuclease,
00:05:44.14 which degrades the RNA following decapping.
00:05:46.26 In some metazoan cells, microRNA components can also be present in P-bodies,
00:05:52.17 or in a related structure, referred to as a GW-body,
00:05:56.19 which is similar to P-bodies and can show some overlap.
00:06:01.03
00:06:03.21 Now P-bodies are proportional to the pool of untranslated RNAs,
00:06:07.09 and I just want to illustrate this, showing the dynamics of these structures.
00:06:11.15 So this is looking at yeast cells during mid-log growth.
00:06:15.02 And you can see there is a few P-bodies of moderate size,
00:06:17.10 in those cells. However, if we starve those cells for glucose,
00:06:21.13 this leads to a rapid loss of translation, and you can see that the P-bodies
00:06:25.18 get quite a bit larger. Conversely, if we treat cells with cyclohexamide,
00:06:31.09 which traps mRNAs in polysomes,
00:06:33.16 that it is in association with ribosomes, the P-bodies disappear.
00:06:37.15 And these type of observations are some of the data which has lead to the model
00:06:43.07 that RNAs partition between translation, or P-bodies, depending upon on whether
00:06:48.08 they are associated with ribosomes or with these components of P-body structures.
00:06:55.00 P-bodies also contain RNA, and we can detect that either by in situ hybridization,
00:07:00.07 or by a common technique which is using the ability to tether GFP to a specific RNA,
00:07:06.14 through sequence specific RNA-binding proteins.
00:07:09.00 Essentially making a GFP-tagged RNA molecule.
00:07:11.13 And if you do that, and express that in cells which are happily growing,
00:07:16.13 here, the RNA tends to be distributed around the cytoplasm,
00:07:19.28 because it is engaged in translation.
00:07:22.06 And we can see that because this is what is called a polysome trace,
00:07:25.07 where the larger of these series of peaks here, these are RNA associated with ribosomes.
00:07:29.26 The larger this peak is here, the more translation that is going on in the cell.
00:07:33.10 Now if we starve that cell for a few minutes with glucose,
00:07:36.06 you can see that translation declines dramatically, these polysomes are now all gone.
00:07:40.22 And now you can see these mRNAs accumulate
00:07:43.15 in these discrete foci within the cell. And those foci overlap with markers of P-bodies.
00:07:48.09 So P-bodies contain RNAs, and those RNAs can actually
00:07:51.21 reversibly leave P-bodies and re-enter translation.
00:07:55.29 Through a number of different experiments
00:07:57.17 shown by Muriel McGees and Daniella Texiera in my lab several years ago.
00:08:02.25 And you can see that if you add glucose back,
00:08:04.17 these P-bodies shrink back down and the polysomes are restored.
00:08:09.01
00:08:11.15 P-bodies don't only contain RNA, but they also require RNA for formation.
00:08:16.02 Here is an experiment done by Marco Valencia Antonio Sanchez in my lab a few years ago,
00:08:22.01 where he purified P-bodies by differential centrifugation,
00:08:25.01 and then if he treated those P-bodies with RNase, you can see that they fell apart.
00:08:29.17 And so P-bodies not only...so P-bodies are complexes that form on non-translating RNA,
00:08:36.11 that contain this discrete set of proteins, and they require that RNA for their formation.
00:08:43.22 So one of the questions my lab has been interested in, is trying to understand,
00:08:46.21 how do these proteins actually assemble onto the RNA,
00:08:50.11 and how do they assemble into a higher order P-body.
00:08:52.06 And what does that tell us about the function of these complexes.
00:08:54.26 And so one approach we have been taking to this,
00:08:57.09 is to actually purify all these different components,
00:08:59.15 and then test for their interactions between them.
00:09:02.10 And we have done a number of different experiments where we purify
00:09:06.00 common versions of these core components, and then test their binding in vitro.
00:09:10.20 For example here, we're taking two different proteins and mix them,
00:09:14.07 we purify this Dhh1 protein back out,
00:09:17.28 and we ask if this one comes along.
00:09:19.16 And if you do a lot of this kind of co-immunoprecipitation experiments,
00:09:22.14 which you can see, is there is a tremendous number of interactions between
00:09:27.08 these core P-body components, and with each other.
00:09:30.07 And with the RNA molecule.
00:09:33.13 So, this cartoon is showing all these components, using purified proteins,
00:09:39.09 showing the direct protein-protein interactions that occur,
00:09:42.15 between these different core components of P-bodies.
00:09:45.13 And what you can see is, there is a dense network of interactions.
00:09:48.02 And within that, many of these proteins are also RNA binding proteins,
00:09:52.09 and so that these proteins can not only assemble with each other,
00:09:54.25 they can also then bind to the RNA molecule to make an RNA-protein complex.
00:09:59.10
00:10:03.00 Now, we don't really know how those come together to form the definitive complex yet.
00:10:09.00 That's one of our goals in the next few years.
00:10:12.05 But, we currently have two models for what these structures look like.
00:10:16.03 One is what we called the closed loop.
00:10:19.00 where the 5-prime and 3-prime end of the RNA are brought together
00:10:21.21 by protein-protein interactions with these different core components,
00:10:25.07 being preferentially bound to the cap, or the 3-prime end of the mRNA.
00:10:29.00 And this kind of closed-loop model is analogous to models for translation complexes,
00:10:34.07 where the cap and poly-A tail are brought together by protein-protein interactions,
00:10:38.15 initiation factors bound to those two sites, to promote the loading of ribosomes.
00:10:45.02 The other possible model is what we call a nucleosome-like,
00:10:48.19 or beads on a string, where we have this core complex, and it's found in multiple places on the RNA,
00:10:54.16 and so the coding region will be coated as well with these different proteins,
00:10:58.27 and current experiments in my lab our directed at trying to identify the differences, to determine
00:11:04.17 which of these complexes actually forms on the RNA.
00:11:09.05
00:11:12.08 Now we understand how these RNAs come together into higher-order structures,
00:11:15.05 from our analysis of these interactions between these proteins.
00:11:19.00 So there are these P-body mRNPs that come together to form a larger structure,
00:11:24.28 through two self interaction domains.
00:11:26.23 So one of these interaction domains is on the EDC3 protein,
00:11:30.18 which can actually dimerize with itself, and so if you disrupt that interaction,
00:11:36.00 what you can see is that you lose...the number of P-bodies goes down dramatically.
00:11:40.16 Although you can still form a few.
00:11:42.08 So this would be a wild-type cell, where we have starved it to make really big P-bodies,
00:11:46.04 and now when we remove that EDC3 protein, you can see there are still some formed.
00:11:51.11 But the ones that formed are dependent on another interaction, which is in the LSM-4 protein,
00:11:56.13 which at its C-terminal tail contains what's called a "prion" domain.
00:12:00.10 OK. And a prion domain is analogous to what we think about in Mad Cow disease,
00:12:05.19 or human Kuru disease, that is a self assembly domain,
00:12:09.12 which at least in the disease state, can be irreversible.
00:12:13.09 But, this is an example where these types of prion domains not only form an aggregate,
00:12:21.02 but that is actually reversible assembly, so it's not a pathogenic state.
00:12:24.20 And if you get both of the EDC3 protein and the prion domain on the LSM4 protein,
00:12:30.15 down here, you see that there are no P-bodies forming anymore.
00:12:34.03 Now, is that interesting that there is actually this prion domain,
00:12:39.25 or what is often called a Q/N domain involved in aggregation of these RNA-protein complexes?
00:12:44.26 I just want to make a few points about this,
00:12:47.19 because I think it is actually a very interesting aspect of the biology,
00:12:50.20 which might have broader significance.
00:12:52.16 First, many proteins involved in translation and RNA-degradation
00:12:57.22 have these type of Q/N domains.
00:12:59.21 For example, if you search the yeast genome,
00:13:02.26 there are about 100 proteins that have the potential
00:13:05.23 to form these type of aggregates through the Q/N or prion domain.
00:13:10.10 Of those 100, 50 are involved in translation or RNA-degradation.
00:13:15.20 So over half. And many of the others we don't know what they do,
00:13:18.11 so they might also be involved.
00:13:20.03 So I think this is going to be a common feature of many of these proteins,
00:13:23.21 in RNA translation and degradation
00:13:26.16 and suggests that they may then have a tendency to form these type of aggregates,
00:13:31.04 for biological reasons we don't understand yet.
00:13:33.09
00:13:35.24 Consistent with that, different types of these domains
00:13:39.13 may create different types of structures, or different types of P-bodies, for example.
00:13:45.17 And, it is not well understood how specific these different Q/N domains can be,
00:13:52.09 but in some cases it appears they can have specific interactions with each other,
00:13:57.08 and in other cases general. So what that means is different types of Q/N domains
00:14:00.28 then could also drive different types of domains, or types of RNA-protein granules.
00:14:05.17 And consistent with that, we also know that Q/N domains
00:14:08.07 are involved in the assembly of another
00:14:10.11 RNA-protein granule in yeast and in human cells called a stress granule.
00:14:14.19 Which I'll discuss in a few minutes.
00:14:16.11
00:14:19.00 Finally, the fact that Q/N domains can be irreversible,
00:14:22.23 allows them to function as epigenetic elements. And what that means then,
00:14:26.23 is that if you assemble RNA-protein granules through these prion domains,
00:14:32.00 you have the potential then to have an heritable genetic state, because of that.
00:14:37.18 And so, a model from Kazeksai and Eric Kandel has proposed that such type of prion driven assembly
00:14:46.05 may play a role in synaptic plasticity in memory formation.
00:14:49.20
00:14:52.17 And then finally, it is striking that several of the neuro-degenerative diseases
00:14:57.16 involve the expansion of poly-glutamine
00:15:01.14 tracts in proteins which are normally components of P-bodies or stress granules.
00:15:05.04 So for example, Huntington's:
00:15:08.01 the protein which gives rise to the neuro-degenerative disease has a polyQ tract in it
00:15:16.14 and it is normally a component of P-bodies. And that is probably why it has the polyQ tract,
00:15:21.06 because part of its biology requires it to assemble into that complex,
00:15:25.04 but when that tract expands too big, it leads to the formation of cytoplasmic aggregates.
00:15:31.09 And it's controversial as to whether those are toxic or protective,
00:15:34.21 but there is a striking correlation between some of these neuro-degenerative diseases,
00:15:38.18 having expansions of these tracts, and the formation of these aggregates,
00:15:43.08 which are probably related in some manner to P-bodies and/or stress granules.
00:15:50.00
00:15:54.16 Alright. So, we understand somewhat then how these P-bodies assemble.
00:16:00.03 And we understand how they aggregate into larger structures.
00:16:03.10 So, this allows us to do an experiment, then to test what is the significance
00:16:08.01 of making these larger structures. Is this larger structure important
00:16:12.24 for these RNAs to stop translation, or is it important for them to be degraded?
00:16:16.24 So, the experiment we have done then, is to simply look at what happens, to RNA-degradation
00:16:24.19 in a mutant that can no longer assemble these larger structures.
00:16:27.25
00:16:29.21 And what we observed then,
00:16:31.16 if we look at degradation of RNAs, is that RNAs tend to degrade pretty normally.
00:16:36.13 So here we are looking at specific reporter mRNA MFA2.
00:16:40.11 We block transcription at 0 time,
00:16:42.06 and you can see that in wild-type cells the RNA degrades with a half life of about 3 minutes.
00:16:46.22 And in the mutant, which can't make large P-bodies, we see a similar decay rate.
00:16:52.08
00:16:55.09 So in other words, formation of these larger scale structures is not required,
00:16:59.17 at least for the degradation of a few reporter mRNAs.
00:17:03.01 And so, that suggests that the formation of these individual mRNPs,
00:17:07.02 at least under the conditions that we have examined so far,
00:17:10.10 is sufficient for allowing RNAs to be degraded, for allowing them to exit translation,
00:17:16.03 and enter this translationally repressed state. So this raises the question then,
00:17:21.27 why do cells make these larger structures?
00:17:25.12 In fact, one has to anticipate that these structures have roles,
00:17:29.24 because they are conserved throughout eukaryotic cells that have been looked at.
00:17:33.16 So, why make these larger structures?
00:17:36.04 The simplest explanation is that you make these larger structures to promote interactions,
00:17:43.15 between components when those components are limiting.
00:17:45.20 And it is very likely under the conditions that we have examined so far in the lab,
00:17:49.29 the components that we are testing, under the conditions that we are examining in the lab,
00:17:56.00 the key components that trigger RNA-degradation, or translation repression, are not limiting.
00:18:02.09 Alternatively, it could be that some mRNAs require aggregation for their regulation.
00:18:07.28 It could be that you aggregate to protect against, to limit other interactions.
00:18:13.13 For example, aggregation of RNA into these structures might protect them from other nucleases,
00:18:18.03 which are not present in those. And then, it could be that aggregation plays important roles
00:18:24.08 in cellular organization, and or transport. As we have suggested in neurons or in germ cells.
00:18:32.10 But this is actually an ongoing area of interest, trying to understand the larger role of assembly,
00:18:40.09 of these large structures in eukaryotic cells. And I want to point out,
00:18:44.20 that this is not a problem that is limited to the study of P-bodies and
00:18:48.14 the regulation of translation in the cytoplasm.
00:18:50.21 In fact, as we have studied more about the organization of cells,
00:18:54.24 what we have learned is that there is a diversity of RNA-protein granules,
00:18:58.08 that form in eukaryotic cells.
00:19:01.04 So, in the cytoplasm we have talked about P-bodies, and a little bit about stress granules.
00:19:05.18 But in the nucleus, there are a lots of other RNA-protein complexes.
00:19:09.15 Nuclear speckles which contain splicing factors, and also Cajal bodies,
00:19:15.02 which contain components of snRNA, small nuclear RNA protein complexes.
00:19:21.04 And actually the work of Carl Neuberger has really shown that the function of these Cajal bodies,
00:19:25.17 is to increase the rate of the assembly of these protein RNA-complexes.
00:19:30.06 And so I think, that one think we should expect as we go forward,
00:19:33.17 as we study these different RNA-protein complexes,
00:19:37.23 is that their general role might be to increase the assembly rates
00:19:42.00 of components within them, simply by providing higher level concentration of those factors.
00:19:49.26
00:19:50.24 Now, P-bodies often dock, or overlap, with these RNA-granules referred to as stress granules.
00:19:59.06 So here, we are looking at a mammalian cell.
00:20:01.15 The blue represents a stress granule structure.
00:20:04.18 And the yellow or red here, represents a P-body.
00:20:08.19 And you can see they are often docked together, near each other.
00:20:11.08 A similar phenomenon occurs in yeast,
00:20:13.12 although in that case, and in many cases, the P-body and stress granule actually overlap.
00:20:19.04 So here, in this particular image in yeast, P-bodies would be red
00:20:23.11 and stress granules would be green.
00:20:26.04 So if they are overlapped, the will be yellow.
00:20:27.16 And you can see that many of these components are in fact yellow.
00:20:30.10
00:20:32.08 So, what are stress granules?
00:20:34.18 So stress granules, again, are an RNA-protein complex containing untranslating RNAs.
00:20:39.21 But in contrast to the decay, and translation repressors present in P-bodies,
00:20:44.24 stress granules contain translation initiation factors,
00:20:47.22 RNA-binding proteins, and, in some cases, they can contain the 40s subunit.
00:20:54.17 Stress granules form when translation initiation is slow,
00:20:58.23 and so the simplest model is that these stress granules represent a population of mRNAs,
00:21:05.16 which are entering or exiting translation.
00:21:07.24 And that is they have assembled a translation initiation complex,
00:21:11.04 but they haven't entered the elongation phase of translation.
00:21:16.16 It's not known whether they are really entering or exiting translation,
00:21:20.02 although in some cases I'm going to argue that they are perhaps entering translation.
00:21:25.01
00:21:27.20 Now, these interactions between P-bodies and stress granules,
00:21:31.06 have lead to the suggestion that mRNAs exchange between these two.
00:21:34.29 And so the logic here is that, while these two different granules interact,
00:21:39.03 they can contain the same mRNA,
00:21:42.28 and we know that mRNAs which are in P-bodies can return to translation.
00:21:46.24 So at some level, those mRNAs must be able to re-associate with translation initiation factors.
00:21:51.20 And so one hypothesis then,
00:21:54.04 is that mRNAs are exchanging between stress granules, and P-bodies.
00:21:57.28
00:22:00.00 And there is really two models for how this could be occurring.
00:22:03.12 So in the first model, mRNAs, when they are engaged in translation,
00:22:10.15 when they cease translation they assemble into this stress granule structure.
00:22:14.08 And within this stress granule, different mRNAs would assemble different complexes.
00:22:19.06 Some RNAs would be targeted for degradation and would be sent to a P-body,
00:22:22.29 for destruction.
00:22:24.14 And other RNAs would reassemble a new translation complex and return to polysomes.
00:22:28.20 The other possibility is, in fact, RNAs when they exit translation they travel to a P-body,
00:22:36.18 and within that P-body some RNAs would be sorted for destruction.
00:22:40.19 Or other RNAs, presumably due to their composition or sequence features,
00:22:45.05 would reassemble a new translation complex,
00:22:47.26 and then would return back to the translated pool.
00:22:50.15 A few years ago, Ross Buck in my lab wanted to try to distinguish between these two models,
00:22:56.28 and so it's relatively a simple experiment,
00:22:59.19 cause he is going to analyze how defects in the ability to assemble P-bodies or stress granules
00:23:04.08 affect the other structures.
00:23:06.16 So for example in this model, you would have to assemble a stress granule
00:23:11.13 in order to make a P-body. Where as in this model, you might have to make a P-body
00:23:15.22 in order to make a stress granule.
00:23:17.02 And so, here's what those kinds of experiments look like.
00:23:21.09 Here we are using yeast cells,
00:23:23.00 where we have a mutation which can prevent the formation of either stress granules or P-bodies.
00:23:27.16 And this is a wild type cell, and again you can see lots of stress granules in green,
00:23:32.13 many P-bodies in red. And they are generally overlapping the stress granule markers,
00:23:37.05 so they are yellow.
00:23:38.16 If you get rid of the ability to form stress granules,
00:23:42.04 you still make P-bodies, and you make about the same number,
00:23:46.02 and the same brightness and the same size of P-bodies.
00:23:51.03 So, mutations reducing stress granules do not affect the formation of P-bodies,
00:23:55.27 at least in yeast cells.
00:23:57.16 However, if we do the converse experiment, where we get rid of P-bodies,
00:24:02.26 and here we are using those mutations we talked about earlier,
00:24:05.10 the EDC3 protein gone and deleting the prion domain on this Lsm4 protein.
00:24:10.16 Now what you see, is that we don't make any P-bodies,
00:24:14.00 but we also don't make any stress granules.
00:24:15.17
00:24:17.04 So our interpretation of that, is in fact, that stress granules form,
00:24:22.07 from pre-existing P-bodies. Now of course, there is two possible views of this:
00:24:28.14 one view is, in fact, that this effect is really an indirect effect.
00:24:32.25 But if you get rid of P-bodies, you have all kinds of changes in the regulation of mRNAs,
00:24:37.13 and that changes the proteins which affect stress granule formation.
00:24:41.03 And therefore you can't make stress granules for some indirect reason.
00:24:44.09 And the other model, is that there in fact, that P-bodies provide an assembly site,
00:24:49.12 where in this model, when RNAs stop translating,
00:24:53.27 they first assemble into these complexes that aggregate in P-bodies, and that with time,
00:24:59.24 some of these RNAs are targeted for translation,
00:25:03.08 so they assemble new translation factors on them,
00:25:06.18 and some are targeted for degradation, and they might go away,
00:25:08.26 and so in this intermediate time we kind of have an overlap,
00:25:12.07 and then with more time, these RNAs are being destroyed, are gone,
00:25:16.26 and these RNAs, which are going to re-enter translation,
00:25:20.06 will become a greater component of the total pool.
00:25:23.23 So in order to look at this, distinguish these possibilities,
00:25:28.18 Ross did an experiment where he basically looked to see if stress granules form
00:25:36.20 by maturation of P-bodies. And so what he is going to do is just follow a time course
00:25:40.29 of the formation of P-bodies and stress granules during a glucose deprivation.
00:25:45.26 So he is going to grow a culture, he's going to starve it for glucose for a short period of time,
00:25:50.13 and then take images over a variety of time,
00:25:53.24 and see what happens to P-bodies and stress granules.
00:25:56.01 And so when he does that experiment, these are the results:
00:26:00.12 The important observations, I'm just going to highlight.
00:26:03.26 The first is, that the first thing you see is that P-bodies get very big.
00:26:08.01 So, from zero time here is before the stress,
00:26:11.27 there are some small P-bodies you can't see under these kind of exposure conditions.
00:26:15.09 But by seven minutes of stress they are quite large,
00:26:18.01 and very bright.
00:26:20.05 So P-bodies form first.
00:26:21.29 The second thing you see, is that the first stress granules you can see,
00:26:26.04 there is a few of them here at this seven-minute time-point,
00:26:29.13 they are always in association with a P-body.
00:26:32.14 So if you look down here in the merge, they always overlap.
00:26:35.04 So stress granules first appear in conjunction with a P-body.
00:26:39.21 And then the third thing you see, is if you follow with more time,
00:26:43.27 some of these P-bodies mature from being a P-body to being primarily like a stress granule.
00:26:50.08 So if you look at this one here, at the early time points, it's primarily a P-body
00:26:54.15 with very little stress granule marker. And then by half an hour later,
00:26:58.18 the amount of Dcp2, a P-body marker there has declined dramatically,
00:27:03.15 and the amount of poly-A binding protein, a stress granule marker has increased,
00:27:07.27 suggesting that these, in fact, are maturing from a P-body state into a stress granule state.
00:27:13.02 And that suggests that there is really a directionality in the movement of RNAs,
00:27:18.29 between these different types of complexes. That they go from translation,
00:27:23.14 to a repressed state, generally in the formation of a P-body, type of complex.
00:27:30.24 And those RNAs can then re-enter translation by forming a new translation complex,
00:27:35.09 which can associate into these larger structures referred to as stress granules,
00:27:39.07 and go back into translation. Although I'm focusing on these individual RNA-protein complexes,
00:27:47.11 we can observe that in the microscope really is a summation of that total population
00:27:52.00 is in these aggregates that are visible in the light microscope.
00:27:56.13
00:28:00.23 Now, so what then are stress granules?
00:28:03.14 So at least under these kind of conditions,
00:28:06.06 this suggest that stress granules are primarily RNAs,
00:28:09.12 which are being primed for re-entering translation.
00:28:12.00 And that then, maybe stress granules should be thought of as sites of assembly,
00:28:18.07 of translation initiation complexes.
00:28:20.06 And the argument here for this is that they form when initiation is limiting,
00:28:24.04 they form from non-translating RNAs, and they contain translation factors.
00:28:28.25 And so maybe they are not necessarily sites where RNAs are targeted for repression,
00:28:32.16 but they are sites where RNAs have their high local concentration of translation factors,
00:28:38.00 and therefore allow for the efficient assembly of these translation initiation complexes
00:28:42.05 which can then go on and enter translation.
00:28:44.17 And this is an area of further research in my lab, as well as other labs, as well.
00:28:50.05
00:28:55.09 Now, I want to step back and just say a few words about RNA granules.
00:28:59.22 Because those of you who work in this area, or read about it,
00:29:04.19 it's easy to become confused about the diversity of different types of RNA granules,
00:29:09.06 which are described in the literature.
00:29:10.24 And, what I'd like to argue is in fact, that maybe these granules are all related,
00:29:16.03 in a simple kind of model where there are different stages
00:29:19.04 of movements of RNAs through this mRNA cycle.
00:29:24.08 Now, so for example, under some conditions you can see granules that accumulate,
00:29:32.25 which contain EIF4e, EIF4g, the cap-binding complex, and poly-A binding protein,
00:29:37.25 that are missing other components,
00:29:40.12 like 40s subunits which form under a different stress.
00:29:44.13 So, a simple way of thinking about these, is that they are simply blocked,
00:29:49.12 they simply have different rate limiting steps in the transitions
00:29:52.13 between these different pools in the mRNA cycle.
00:29:55.09 So if you block this step here,
00:29:57.04 in response to a various cue, these RNAs accumulate with these complexes,
00:30:01.25 then you will get a stress granule of that composition. Whereas if you block over here,
00:30:05.28 you'll accumulate with this type of RNA-protein complex,
00:30:09.02 and you'll get a stress granule which contains these other factors.
00:30:12.06 So, these aren't fundamentally different particles perhaps,
00:30:17.07 they are simply different stall points on a continuum of exchange points.
00:30:22.21 And so when one thinks about that kind of model then,
00:30:26.19 the diversity and the composition of granules observed in the cell
00:30:30.23 can also be affected by the behavior of different mRNA sub-populations.
00:30:36.06 So for example, under some stresses you get both P-body increasing,
00:30:42.20 and stress granules. But probably because there are some mRNAs which are stalled at this stage,
00:30:48.21 and they are assembled with these P-body components, you see a large formation of P-bodies,
00:30:54.01 but other mRNAs are stalled at a different site.
00:30:56.09 Perhaps over here after they have a small subunit,
00:31:00.11 and so you will also see stress granules accumulate.
00:31:02.22 And so one of the things we don't understand very well,
00:31:05.04 is what is the diversity of different types of mRNAs, and mRNA protein complexes,
00:31:09.12 and then how they lead to the accumulation of different particles under different conditions.
00:31:17.15
00:31:19.16 But one thing consistent with this, is if we look in the literature,
00:31:23.15 what we can see is that there is really a continuum of RNA-protein granules,
00:31:27.07 which span from a classical P-body as defined by the initial papers, to a classical stress granule.
00:31:34.19 And so this is just a cartoon of that, and the important thing to look at here,
00:31:38.26 is underneath each of these particles, is shown a list of the proteins found in them,
00:31:44.09 and in green would be components which are only seen in P-bodies.
00:31:47.28 So this would be a P-body which has only components of P-bodies.
00:31:51.13 Yellow are proteins which can be seen in either P-bodies or stress granules.
00:31:55.21 And red would be components which are only seen in stress granules.
00:31:59.11 And if you look around the literature, dendritic P-bodies,
00:32:03.16 these are RNA-protein complexes found
00:32:06.10 at the dendritic side of synapses in various neurons.
00:32:10.08 They have a mixture of both P-bodies and stress granule components.
00:32:14.10 Transport particles in neurons in Drosophila have a mixture again.
00:32:21.10 In C. elegans, different types of granules can again have a mixture.
00:32:25.11 Although in many cases we do not know the complete composition of each of these granules,
00:32:29.12 what you begin to see is that there is no such thing as, there are two boundary conditions,
00:32:35.02 but then there is a wide range of different intermediate forms.
00:32:39.14 And that we should perhaps think of these then,
00:32:42.25 all as different stall points on this mRNA cycle,
00:32:45.22 or different sub-populations of mRNAs stalled at a mixture of sites.
00:32:49.25
00:32:51.24 Alright. So in summary then, I just want to try to highlight the main points that I've tried to say
00:32:58.16 which is that, in cells, mRNAs can exist in at least two predominate mRNP states;
00:33:04.12 those that are translating, and those that are repressed.
00:33:07.13 Those repressed mRNAs typically accumulate in RNA-protein granules,
00:33:11.11 such as P-bodies and stress granules.
00:33:13.21 These different biochemical compartments, can have different rates of translation,
00:33:20.08 deadenylation, and mRNA degradation, depending upon where the mRNA is in the cell,
00:33:25.22 and what proteins it is associated with.
00:33:27.07 P-bodies may also be related to RNA transport particles,
00:33:31.09 and therefore play some role in localization,
00:33:33.02 and that there are mechanisms which move RNAs between these compartments,
00:33:36.24 which I haven't had time to talk about today,
00:33:39.03 which are discreet and involve various types of proteins and enzymes.
00:33:43.15
00:33:45.20 There are lots of unanswered questions in this area.
00:33:48.15 How do mRNAs actually cease translation and assemble into P-bodies?
00:33:52.19 How do mRNAs get out of P-bodies and reassemble a new translation complex?
00:33:56.25 And get back into the translating pool?
00:33:59.05 How do they determine which mRNAs are going to do which?
00:34:01.23 And why do you actually form these large scale aggregates?
00:34:05.03 The prediction is, to promote interactions between limiting components,
00:34:08.21 but there is no direct demonstration of that for P-bodies or stress granules yet.
00:34:14.16 And then, how does this cycle relate to the localization of mRNAs to certain regions of the cell
00:34:19.15 and the biogenesis of mRNAs in the nucleus,
00:34:21.26 and their entry into the cytoplasm and beginning to translate and function?
00:34:26.18 And with that, I'd like to thank you all for your attention.
00:34:30.29

Videos in this Talk
  • Part 1: mRNA Localization, Translation and Degradation
    Part 1: mRNA Localization, Translation and Degradation
    Audience:
    • Researcher
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
  • Part 2: P-bodies and the mRNA Cycle
    Part 2: P-bodies and the mRNA Cycle
    Audience:
    • Researcher
    • Educators of Adv. Undergrad / Grad
Speaker: Roy Parker
Total Duration: 1:28:40
Recorded: February 2014
All Talks in Genetics and Gene Regulation

Talk Overview

The control of eukaryotic mRNA production and function is a key aspect of the regulation of gene expression. In the first part of this lecture, I will discuss how in eukaryotic cells, the control of mRNA localization, translation and degradation in the cytoplasm allow for the proper regulation of the amount, duration, and location of protein production. The basic mechanisms of these processes are understood and reveal that the mechanisms of localization, translation, and degradation are interconnected. The unique properties of each mRNA are dictated by its intrinsic interactions with cellular machines, as well as its complement of mRNA specific RNA binding proteins and miRNAs. Strikingly, mRNPs are dynamic and can be modulated by protein modifications as well as by modification of the mRNA itself, thereby providing a diversity of targets for the regulation of mRNA function in response to extracellular signals.

In the second part of this lecture, I will provide an overview of why the regulation of translation and mRNA degradation is an important aspect of the control of gene expression in eukaryotic cells. In addition to the translating pool of mRNAs associated with polysomes, recent experiments have identified P-bodies and stress granules as specific cytoplasmic compartments wherein untranslated mRNAs accumulate. In addition to mRNAs, P-bodies tend to contain translation repressors and mRNA degradative enzymes, while stress granules reflect mRNAs in association with some translation initiation factors and RNA binding proteins. P-bodies and stress granules interact and suggest a dynamic process wherein eukaryotic mRNAs remodel their interacting proteins and enter and exit translation, thereby affecting the control of mRNAs in the cytoplasm. We are interested in defining the mechanisms by which P-bodies and stress granules assemble and how cells regulate the movement of mRNAs between these different biochemical and cell biological compartments. Several approaches will be described including biochemical and genetic analyses of known proteins modulating these events, as well as the identification of new factors affecting P-body and stress granule formation and function.

Speaker Bio

Roy Parker

Roy Parker

Roy Parker a professor of chemistry and biochemistry and Cech-Leinwand Endowed Chair of Biochemistry at the University of Colorado, Boulder and an Investigator of the Howard Hughes Medical Institute. Prior to joining the University of Colorado in 2012, Parker was a Professor of Molecular and Cellular Biology at the University of Arizona. Dr. Parker received… Continue Reading

Playlist: RNA Processing

Related Resources

Buchan JR, Parker R. Eukaryotic stress granules: the ins and outs of translation. Mol Cell. 2009 Dec 25;36(6):932-41.

Balagopal V, Parker R. Polysomes, P bodies and stress granules: states and fates of eukaryotic mRNAs. Curr Opin Cell Biol. 2009 Jun;21(3):403-8.

Franks TM, Lykke-Andersen J. The control of mRNA decapping and P-body formation. Mol Cell. 2008 Dec 5;32(5):605-15.

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