Discovery Talk Questions and Answers: Discovering Ribozymes

Tom Cech

Questions

In his talk, Dr. Cech refers to intron sequences. How are they referred to in his 1981 Cell paper?
1. Internal nucleic acid sequences
2. Intragenic virtual sequences
3. Intervening sequences
4. Inteins
5. None of the above
In his talk, Dr. Cech states that he found in his studies of transcription that “RNA splicing was taking place outside of the cell, in vitro, with our purified extracted RNA” (1:41).
Using evidence from the 1981 paper, which of the following statements would you say is true?
1. Preliminary results showed that rRNA splicing could be completed in isolated nuclei, hinting that splicing probably involved enzymatic proteins.
2. Preliminary results showed that rRNA splicing could be completed in a transcription solution, using purified rRNA templates, hinting that splicing did not require enzymatic proteins.
3. None of the above
In his talk, Dr. Cech says that he found that “Initially, this splicing reaction was taking place in the same cocktail of small molecules that were necessary for transcription” (2:03).
3.A. Using the 1981 paper, which of the following statements would you say correctly describe(s) what Dr. Cech and his team were trying to do when they made this observation (choose all that apply)?
1. They were trying to demonstrate that pre-rRNA was self-splicing
2. They were trying to synthesize pre-rRNA in vitro to then develop a splicing assay, using purified pre-rRNA as a substrate
3. They were planning on identifying proteins from the nuclear extract that could be involved in RNA splicing using purified pre-rRNA as a substrate
4. None of the above
3.B. Explain why this observation later led the team to hypothesize an autocatalytic activity for rRNA.
In his talk, Dr. Cech also explains that he “decided that it would be useful to see which of those small molecules was really required, not for the transcription part, but for the RNA splicing. So I added them, I subtracted them one at a time from the transcription cocktail, I added them back (2:13).” What were these molecules (choose all that apply)?
1. GTP
2. MgCl2
3. (NH4)2SO4
4. Pre-rRNA
5. None of the above
Dr. Cech states that, for excision of the IVS to take place, “G was required, A wouldn’t do it, C wouldn’t do it, U wouldn’t do it. It had to be guanosine (2:46).”Discuss how the supporting evidence for this claim was presented in the paper.
When he found that G was a required ingredient in the splicing reaction, Dr. Cech explains in his talk that he “didn’t really know what to make of it.” He also states that this data was obtained before Art Zaug “found that the sequence of the cut out RNA started with a guanosine residue”(3:01) and Joe Gall’s laboratory “had sequenced the gene that encodes this RNA” and had found “that there was no guanine present near that exon-intron boundary” (3:27).
Compare the actual timeline of the scientific discovery as described in the talk with the way it was presented to other scientists through the peer-reviewed journal.
When describing the key experiment in his discovery, Dr. Cech explains that when he added “in one tube, radiolabeled GTP to the purified precursor RNA, and in other tubes, the other nucleotides (…), only in the lane where I had added the radiolabeled GTP was there a radiolabeled RNA band the exact size of the intervening sequence.” (4:53)
Describe how this data was presented in the paper.
Comparing the RNA splicing models presented in Dr. Cech’s talk and his paper, which step is described in the paper but not in the talk?
Why did Dr. Cech, despite evidence that denaturing treatments did not destroy splicing activity, believe that an enzyme should be responsible for the splicing activity?
In the 1981 paper, what experiment(s) do the authors suggest could be done to test for the involvement of a protein in the splicing reaction (choose all that apply)? [paper]
1. Subject the splicing intermediate to boiling in the presence of SDS and 2-mercaptoethanol to destroy association with proteins
2. Use the SDS-phenol method to extract pre-rRNA to make sure it is not bound to proteins
3. Detect a protein bound to the splicing intermediate by using radioactive amino acid labeling
4. Test exon ligation by S1-nuclease-protection studies of hybrids formed between the in vitro splicing product and the rDNA
5. None of the above

Answers

c- Intervening sequences (IVS).
p487: “Many eucaryotic tRNA, rRNA and mRNA genes are interrupted by stretches of noncoding DNA called intervening sequences (IVSs), which are transcribed as part of a precursor RNA and are subsequently removed by a cleavage-ligation reaction termed splicing.”Wikipedia reference: The word intron is derived from the term intragenic region; i.e., a region inside a gene. http://en.wikipedia.org/wiki/Intron

The term “intervening sequence” was replaced over time by the term “intron,” because it was convenient to talk about the intron-exon structure of genes and of their RNA transcripts.
a
The authors were aware of some sort of splicing activity taking place in vitro, although it could be attributed to enzymes that were found in the nuclei and nucleoli with the RNA.p487: Preliminary studies had shown that “When isolated Tetrahymena nuclei are incubated under conditions that allow elongation of rRNA chains initiated in vivo, the rRNA precursor is synthesized and spliced (Zaug and Cech, 1980). Transcription and splicing also occur in isolated nucleoli (Carin et al., 1980).”
A. b, c
It was hypothesized by Cech et al. that the nuclei contained the unknown factors responsible for catalyzing splicing. The transcription cocktail, on the other hand, was an artificial system that was known to allow transcription and contained clearly identified molecules that were not expected to promote splicing. Dr. Cech was hoping to use the in vitro transcription system to synthesize pure pre-rRNA. The plan was to use the pre-rRNA as a substrate for a splicing assay that would allow the identification of nuclear protein(s) involved in splicing.Fig. 2, p. 488 “We hoped to use the unspliced pre-rRNA synthesized in vitro under low salt conditions as a substrate to assay for splicing activity in nuclear extracts. This approach proved to be impossible, however, because excision of the IVS occurred when the isolated RNA was incubated in transcription cocktail (see Experimental Procedures) at normal salt concentration in the absence of a nuclear extract.”

p. 495: “Transcription and Splicing in Isolated Nuclei: Transcription reactions contained 1 to 2 X 107 nuclei in 0.45 ml of transcription cocktail (53 mM Tris-HCI [PH 8.01, 5.3 mM MgCl2, 1 mM CaCl2, 1 mM putrescine, 1 mM spermidine, 0.1 mM spermine, 2 mM 2-mercaptoethanol, 0.048 mM each of ATP, CTP, UTP and GTP, 0.2-0.4 mM aurintricarboxylic acid (nuclease inhibitor) and 40 ug/ml alpha-amanitin).”

3.B. Because the transcription cocktail consisted of small molecules whose activity was well known, not enzymes, it was clear to the authors that the transcription cocktail by itself was unlikely to be responsible for the splicing observed here. Therefore, this observation led the authors to hypothesize that rRNA could have an autocatalytic activity.

p. 488 – “Inhibition of splicing in isolated nuclei therefore leads to the accumulation of a splicing intermediate that can complete the excision of the IVS under favorable conditions. The activity of this intermediate may be due to a splicing enzyme tightly bound to the pre- rRNA, or it may be a novel case of an RNA-mediated reaction that requires no protein (see Discussion).”
a, b, c, d
Video: “one was a simple salt, magnesium chloride, and the other was one of the precursors of RNA itself, guanosine triphosphate”
Figure 2 and Figure 3 and p488: “Requirements for IVS Excision. Three components of the transcription cocktail (see Experimental Procedures) proved to be necessary to cause excision of the IVS from the splicing intermediate. These components were a monovalent cation, a divalent cation and GTP. We determined the optimum concentration of each cofactor empirically by incubating the splicing intermediate (purified as described in Experimental Procedures) with the three cofactors, two of which were held at fixed concentration while the concentration of the third was varied”
Quote 3: p 489: “Excision of the IVS occurred at a maximal level with ≥75 mM (NH4)2SO4, 5-10 mM MgCl2 and ≥1 uM GTP.”
Although this is an important part of the talk, there is no data in the paper to support this statement. The authors briefly describe the unpublished results they obtained, and indicate that they found that at concentrations below 10uM, none of the nucleotides could replace GTP. At high concentrations (over 1mM), they found some reaction, which they explain as being due to contamination of the nucleotide solution with GTP.p. 489 “We found that ATP, CTP and UTP at concentrations as high as 10 uM did not substitute for GTP in the reaction (data not shown). (Some reaction was observed with 1 mM ATP, CTP or UTP, which could be explained by a 0.01 %-0.10% contamination of GTP in these other nucleotides.)”
In the introduction of the paper, the authors describe the sequencing of the RNA and its corresponding DNA as preliminary data. As Dr. Cech explains in his talk, the specificity of G in IVS excision was not understood initially. This observation alone did not allow the authors to explain the process that took place during IVS excision. However, after the authors sequenced the RNA and compared it to the initial sequence of the DNA, they found that the G must have been added at some point around the excision. In the paper, the authors did not present the facts in a chronological manner but in a logical manner. The data necessary for readers to understand the IVS excision experiments, including sequencing results, was presented as preliminary data.p. 487: “We have recently analyzed the sequence of the excised IVS RNA and have compared it with the sequence of the corresponding region of the rDNA, determined by N. Kan and J. Gall (manuscript in preparation). Both RNAase T1 fingerprint analysis of uniformly labeled IVS RNA and sequence gel analysis of 5’-end-labeled IVS RNA led to the same conclusion: the IVS RNA has a 5’ terminal pG (guanosine 5’- monophosphate) not found in the DNA sequence, and thereafter has a sequence colinear with that of the rDNA (A. Zaug and T. Cech, manuscript in preparation). We now present evidence that excision of the IVS requires a guanosine residue, which is added to the 5’ end of the IVS during excision.”
The evidence shown in the 1981 paper involves experiments with radiolabeled GTP and ATP.p. 489: “When the splicing intermediate (either 3H-labeled or unlabeled) was incubated with (alpha-32P-GTP under the conditions for IVS excision described above, 32P radioactivity was bound only to one RNA species, which had the electrophoretic mobility of the excised linear IVS (Figure 6B). (…) No labeling of the IVS occurred in parallel incubations, in which a-32P-ATP was substituted for the GTP or in which Mg2+ was excluded from the reaction.”
The paper discusses the three steps of IVS excision:
1. G transfer to IVS,
2. IVS release and exon joining
3. Cyclization of IVS
In his talk, Dr. Cech discusses steps 1 and 2 in some detail, but for reasons of brevity, he does not go into IVS cyclization.

p. 493: “According to this model, the splicing enzyme is a phosphoester transferase. The guanosine cofactor provides a free 3’ hydroxyl, to which the 5’ end of the IVS is transferred. A second phosphoester transfer releases the IVS and joins the two exons, while a third transfer cyclizes the IVS and releases the enzyme and the guanosine cofactor.”

Video: (5:36) “this guanine that was added during the splicing reaction was being joined to the 5′ end of the intron. So it looked like it was attacking the splice site phosphate, and forming a new oxygen-phosphorus bond that hadn’t been there before. Now if that happened, that would explain how the GTP would be covalently bound to the end of the intron. But what would happen with the other product of this reaction? Well, the 5′ exon would then have to be released with a hydroxyl group at its 3′ end. And exactly the same kind of chemical step if it occurred now between this exon and the downstream splice site would result in ligation of the exons and release of the intron RNA with this diagnostic guanosine at its 5′ end.”
Because RNA had never been shown to have a catalytic activity, Dr. Cech and his team were hesitating between the two options that were consistent with the knowledge of the time:
1) Non-specific reaction, not mediated by an enzyme
2) Specific reaction, mediated by an enzyme

Video (7:51) “We were assuming that there had to be a protein enzyme that was responsible for a reaction that took place with this incredible specificity, after all in this long RNA there was only this one site that was being chosen as a splice site. There was also specificity with respect to guanosine relative to the other nucleotides. And the reaction was speeded up many billions of fold faster than a spontaneous phosphotransesterification reaction would be predicted to occur. So, if all biological catalysts are protein enzymes, where was the protein?”
p. 492: “The specificity of the reactions performed by the splicing intermediate, and the absolute dependence of these reactions on a guanosine compound, make it seem very likely that an enzyme is associated with the RNA.”
c, d
a, b: These experiments were done previously.
p. 488: “The pre-rRNA isolation procedure included two SDS-phenol extractions, sedimentation in a formamide-sucrose gradient, and in some cases protease treatment”p. 492: “The [splicing] activity is not destroyed or removed, however, when the splicing intermediate is subjected to the following treatments: boiling in the presence of SDS and 2-mercaptoethanol, SDS-phenol extraction at room temperature or at 65°C sedimentation in a 60% formamide sucrose gradient or treatment with proteases according to a variety of protocols described in Experimental Procedures.”

c, d: These were suggested as future experiments in the paper.

p. 492: “Attempts to detect a protein on the splicing intermediate by labeling with radioactive amino acids are in progress.”

p. 492: “Alternatively, the two exons may be held together in the splicing complex without having been ligated. A more direct test of ligation may be provided by S1 -nuclease-protection studies of hybrids formed between the in vitro splicing product and the rDNA.”