55 143 SelfSplicing RNAs
wea25324_ch14_394-435.indd Page 427 13/12/10 7:24 AM user-f467 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile 14.3 Self-Splicing RNAs 427 10-mer P GF (a) 1 Test exon No ESS activity No GFP produced White cells GF P (b) 2 ESS activity GFP produced Green cells Figure 14.40 A reporter construct to detect ESS activity. Burge and colleagues constructed a plasmid containing the two exons of the GFP gene, separated by an intron that held a test exon (red) into which random 10-mers (yellow) had been placed. They transfected cells with collections of these plasmids, and then screened for green color. (a) If the 10-mer has no ESS activity, splicing of the test exon will not be silenced, so it will be included in the middle of the GFP mRNA, disrupting its activity, and producing white cells. (b) If the 10-mer does have ESS activity, the test exon will not be recognized, so it will be spliced out along with the surrounding intron. Thus, a normal GFP mRNA will be produced and the cells will be green. 14.3 Self-Splicing RNAs circular introns (plus a linear intron missing 15 nt), which they had observed in previous studies. This suggested that the RNA was being spliced, and that the excised intron was circularizing. Was this splicing carried out by the RNA itself, or was the RNA polymerase somehow involved? To answer this question, Cech and coworkers ran the RNA polymerase reaction in the presence of polyamines (spermine, spermidine, and putrescine) that inhibit splicing. Then they electrophoresed the products, excised all four RNA bands plus the material that remained at the origin, and purified the RNAs. Next they incubated these RNAs under splicing conditions (no polyamines) and reelectrophoresed them. When they autoradiographed the electrophoretic gel, they could see the intron in the lanes containing RNA from three of the bands. Thus, these bands appear to be 26S rRNA precursors that can splice themselves without any protein, even RNA polymerase. The band we are calling the intron is the right size, but is it really what we think it is? Cech and coworkers sequenced the first 39 nt of this RNA and showed that they corresponded exactly to the first 39 nt of the intron. Therefore, it seemed clear that this RNA really was the intron. Cech’s group also discovered that the linear intron—the RNA we have been discussing so far—can cyclize by itself. One of the most stunning discoveries in molecular biology in the 1980s was that some RNAs could splice themselves without aid from a spliceosome or any other proteins. Thomas Cech (pronounced “Check”) and his coworkers made this discovery in their study of the 26S rRNA gene of the ciliated protozoan, Tetrahymena. This rRNA gene is a bit unusual in that it has an intron, but the thing that really attracted attention when this work was published in 1982 was that the purified 26S rRNA precursor spliced itself in vitro. In fact, this was just the first example of self-splicing RNAs containing introns called group I introns. Subsequent work revealed another class of RNAs containing introns called group II introns, some of whose members are also self-splicing. Group I Introns To make the self-splicing RNA, Cech and coworkers cloned part of the 26S rRNA gene containing the intron, and transcribed it in vitro with E. coli RNA polymerase. When they electrophoresed the labeled products of these transcription reactions, they observed four large RNA products, plus three smaller RNAs corresponding in size to the linear and wea25324_ch14_394-435.indd Page 428 428 13/12/10 7:24 AM user-f467 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile Chapter 14 / RNA Processing I: Splicing All they had to do was raise the temperature, and the Mg21 and salt concentrations, and at least some of the purified linear intron would convert to circular intron. So far, we have seen that the rRNA precursor can remove its intron, but can it splice its exons together? Cech and coworkers used a model splicing reaction to show that it can (Figure 14.41a). They began by cloning a part of the Tetrahymena 26S rRNA gene including 303 bp of the first exon, the whole intron, and 624 bp of the second exon into a vector with a promoter for phage SP6 polymerase. To generate the labeled splicing substrate, they transcribed this DNA in vitro with SP6 polymerase in the presence of [a-32P]ATP. Then they incubated this RNA under splicing conditions with and without GTP and electrophoresed the products. Lane 1 displays the products of the reaction with GTP. The familiar linear intron is present, as well as a small amount of circular intron. In addition, a prominent band (a) (b) E PSP6 E Transcription with [α-32P]ATP +GTP –GTP 1 2 Substrate Ligated exons Circular intron Linear intron Exon1 Intron Exon 2 Splicing substrate GTP + Ligated exons Intron Figure 14.41 Demonstration of exon ligation. (a) Experimental scheme. Cech and coworkers constructed a plasmid containing part of the Tetrahymena 26S rRNA gene: 303 bp of exon 1 (blue); the 413-bp intron (red); and 624 bp of exon 2 (yellow). They linearized the plasmid by cutting it with EcoRI, creating EcoRI ends (E), then transcribed the plasmid in vitro with phage SP6 RNA polymerase and [a-32P]ATP. This yielded the labeled splicing substrate. They incubated this substrate under splicing conditions in the presence or absence of GTP, then electrophoresed the splicing reactions and detected the labeled RNAs by autoradiography. (b) Experimental results. In the presence of GTP (lane 1), a prominent band representing the ligated exons appeared, in addition to bands representing the linear and circular intron. In the absence of GTP (lane 2), only the substrate band appeared. Thus, exon ligation appears to be a part of the self-splicing reaction catalyzed by this RNA. (Source: (b) Inane, T., F.X. Sullivan, and T.R. Cech, Intermolecular exon ligation of the rRNA precursor of Tetrahymena: Oligonucleotides can function as 59-exons. Cell 43 (Dec 1985) f. 1a, p. 432. Reprinted by permission by Elsevier Science.) representing the ligated exons appeared. By contrast, lane 2 shows that no such products appeared in the absence of GTP; only the substrate was present. This is what we expect because splicing of group I introns is dependent on GTP, and it reinforces the conclusion that these products are all the result of splicing. In summary, these data argue strongly for true splicing, including the joining of exons. Cech’s group had already shown that splicing of the 26S rRNA precursor involved addition of a guanine nucleotide at the 59-end of the intron. To verify that selfsplicing in the absence of protein used the same mechanism, they performed a two-part experiment. In the first part, they incubated the splicing precursor with [a-32P]GTP under splicing and nonsplicing conditions, then electrophoresed the products to see if the intron had become labeled. Figure 14.42a shows that it had, and a similar experiment with [g-32P]GTP gave the same results. In the second part, these workers 59-end-labeled the intron with [a-32P]GTP in the same way and sequenced the product. It gave exactly the sequence expected for the linear intron, with an extra G at the 59-end (Figure 14.42b). This G could be removed by RNase T1, demonstrating that it is attached to the end of the intron by a normal 59-39phosphodiester bond. Figure 14.43 presents a model for the splicing of the Tetrahymena 26S rRNA precursor, up to the point of ligating the two exons together and formation of the linear intron. We have seen that the excised intron can cyclize itself. Cech and his coworkers showed that this cyclization actually involves the loss of 15 nt from the 59-end of the linear intron. Three lines of evidence led to this conclusion: (1) When the 59-end of the linear intron is labeled, none of this label appears in the circularized intron. (2) At least two RNase T1 products (actually three) found at the 59-end of the linear intron are missing from the circular intron. (3) Cyclization of the intron is accompanied by the accumulation of an RNA 15-mer that contains the missing RNase T1 products. But this is not the end of the process. After cyclization, the circular intron opens up again at the very same phosphodiester bond that formed the circle in the first place. Then the intron recyclizes by removing four more nucleotides from the 59-end. Finally, the intron opens up at the same bond that just formed, yielding a shortened linear intron. Figure 14.44 presents a detailed mechanism of the cyclization and relinearization of the excised intron. Notice that throughout the splicing process, for every phosphodiester bond that breaks a new one forms. Thus, the free energy change of each step is near zero, so no exogenous source of energy, such as ATP, is required. Another general feature of the process is that the bonds that form to make the circular introns are the same ones that break when the circle opens up again. This tells us that these bonds are special; the threedimensional shape of the RNA must strain these bonds to wea25324_ch14_394-435.indd Page 429 13/12/10 7:24 AM user-f467 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile (a) (b) 1 2 3 4 Origin L IVS 5 G A C U A U U G A (30)A A A G G G A G G U (20)N U C C A U U U A U (10)A A C G A U A 429 Enzyme OH– Phy M U2 T1 OH– 14.3 Self-Splicing RNAs A A G Figure 14.42 Addition of GMP to the 59-end of the excised intron. (a) Radioactive GTP labels the intron during splicing. Cech and coworkers transcribed plasmid pIVS11 under nonsplicing conditions with no labeled nucleotides. They isolated this unlabeled 26S rRNA precursor and incubated it under splicing conditions in the presence of [a-32P]GTP. Then they chromatographed the products on Sephadex G-50, electrophoresed the column fractions, and autoradiographed the gel. Lanes 1–4 are successive fractions from the Sephadex column. Lane 5 is a linear intron marker. Lanes 2 and 3 contain the bulk of the linear intron, and it is labeled, indicating that it had incorporated a labeled guanine nucleotide. (b) Sequence of the make them easiest to break during relinearization. This strain would help to explain the catalytic power of the RNA. At first glance, there appears to be a major difference between the splicing mechanisms of spliceosomal introns and group I introns: Whereas the group I introns use an exogenous nucleotide in the first step of splicing, spliceosomal introns use a nucleotide that is integral to the intron itself. However, on closer examination we see that the difference might not be as great as it seems. Michael Yarus and his colleagues used molecular modeling techniques to predict the lowest energy conformation of the Tetrahymena 26S rRNA intron as it associates with GMP. They proposed that part of the intron folds into a double helix with a pocket that holds the guanine nucleotide through hydrogen bonds (Figure 14.45). This guanine, held fast to the intron, labeled intron. Cech and coworkers used an enzymatic method to sequence the 59-end of the RNA. They cut it with base (OH2 ), which cuts after every nucleotide; RNase Phy M, which cuts after A and U; RNase U2, which cuts after A; and RNase T1, which cuts after G. Treatment of each RNA sample is indicated at top. The deduced sequence is given at left. Note the 59-G at bottom. (Source: Kruger K., P.J. Grabowski, A.J. Zaug, J. Sands, D.E. Gottschling and T.R. Cech, Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell 31 (Nov 1982) f. 4, p. 151. Reprinted by permission of Elsevier Science.) behaves in essentially the same way as the adenine in spliceosomal introns. Of course, it cannot form a lariat because it is not covalently linked to the intron. Until the discovery of self-splicing RNAs, biochemists thought that the catalytic parts of enzymes were made only of protein. Sidney Altman had shown a few years earlier that RNase P, which cleaves extra nucleotides off the 59-ends of tRNA precursors, has an RNA component called M1. But RNase P also has a protein component, which could have held the catalytic activity of the enzyme. In 1983, Altman confirmed that the M1 RNA is the catalytic component of RNase P (Chapter 16). This enzyme and self-splicing RNAs are examples of catalytic RNAs, which we call ribozymes. Actually, the reactions we have seen so far, in which group I introns participate, are not enzymatic in the strict wea25324_ch14_394-435.indd Page 430 430 13/12/10 7:24 AM user-f467 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile Chapter 14 / RNA Processing I: Splicing (a) Pre-rRNA 5.8S begins with an attack by a guanine nucleotide on the 59-splice site, adding the G to the 59-end of the intron, and releasing the first exon. In the second step, the first exon attacks the 39-splice site, ligating the two exons together, and releasing the linear intron. The intron cyclizes twice, losing nucleotides each time, then linearizes for the last time. 26S } 17S Cloned Intron (b) Group II Introns Exon 1 5′ Exon 2 3′ GpU UpA GOH Intron Exon 1 5′ Exon 1 GpA UOH Exon 2 GpU Exon 2 UpU + GpA 3′ Intron GOH Figure 14.43 Self-splicing of Tetrahymena rRNA precursor. (a) Structure of the rRNA precursor, containing the 17S, 5.8S, and 26S sequences. Note the intron within the 26S region (red). The cloned segment used in subsequent experiments is indicated by a bracket. (b) Self-splicing scheme. In the first step (top), a guanine nucleotide attacks the adenine nucleotide at the 59-end of the intron, releasing exon 1 (blue) from the rest of the molecule and generating the hypothetical intermediates shown in brackets. In the second step, exon 1 (blue) attacks exon 2 (yellow), performing the splicing reaction that releases a linear intron (red), and joins the two exons together. Finally, in a series of reactions not shown here, the linear intron loses 19 nt from its 59-end. sense, because the RNA itself changes. A true enzyme is supposed to emerge unchanged at the end of the reaction. But the final linearized group I intron from the Tetrahymena 26S rRNA precursor can act as a true enzyme by adding nucleotides to, and subtracting them from, an oligonucleotide. We should also make another qualification about ribozymes. They can operate on their own in vitro. But many, including many group I introns, are aided by proteins in vivo. These proteins have no catalytic activity of their own, but they can stabilize the catalytically active structure of the ribozyme. As such, these ribonucleoprotein complexes can be called RNPzymes. SUMMARY Group I introns, such as the one in the Tetrahymena 26S rRNA precursor, can be removed in vitro with no help from protein. The reaction The introns of fungal mitochondrial genes were originally classified as group I or group II according to certain conserved sequences they contained. Later, it became clear that mitochondrial and chloroplast genes from many species contained group I and II introns, and that RNAs containing both classes of intron have members that are selfsplicing. However, the mechanisms of splicing used by RNAs with group I and group II introns are different. Whereas the initiating event in group I splicing is attack by an independent guanine nucleotide, the initiating event in group II splicing involves intramolecular attack by an A residue in the intron to form a lariat. The lariat formation by group II introns sounds very similar to the situation in spliceosomal splicing of nuclear mRNA precursors, and the similarity extends to the overall shapes of the RNAs in the spliceosomal complex and of the group II introns, as we saw in Figure 14.20. This implies a similarity in function between the spliceosomal snRNPs and the catalytic part of the group II introns. It may even point to a common evolutionary origin of these RNA species. In fact, it has been proposed that nuclear pre-mRNA introns descended from bacterial group II introns. These bacterial introns presumably got into eukaryotic cells because they inhabited the bacteria that invaded the precursors of modern eukaryotic cells and evolved into mitochondria. This hypothesis has become even more attractive since the discovery of group II introns in archaea, as well as in two classes of bacteria: cyanobacteria and purple bacteria. If we assume that the group II introns are older than the common ancestor of these two bacterial lineages, then they are old enough to have inhabited the bacteria that were the ancestors of modern eukaryotic organelles. Nevertheless, convergent evolution to a common mechanism also remains a possibility. SUMMARY RNAs containing group II introns self- splice by a pathway that uses an A-branched lariat intermediate, just like the spliceosomal lariats. The secondary structures of the splicing complexes involving spliceosomal systems and group II introns are also strikingly similar. wea25324_ch14_394-435.indd Page 431 13/12/10 7:24 AM user-f467 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile Summary Linear intron: 13 nt 5′G 431 UpACCUpUUG OH G 15 nt (1) (2) GpACCUpUUG 19 nt GpUUG C-15 C-19 H2O (3) H2O (5) (4) pACCU pACCUpUUG L-15 OH L-19 OH G Figure 14.44 Fate of the linear intron. We begin with the linear intron originally excised from the 26S rRNA precursor. This can be cyclized in two ways: In reaction 1 (green arrows), the 39-terminal G attacks the bond between U-15 and A-16, removing a 15-nt fragment and giving a circular intron (C-15). In the alternative reaction (2, blue arrows), the terminal G attacks 4 nt farther into the intron, removing a pUUG G 19-nt fragment and leaving a smaller circular intron (C-19). Reaction 3, C-15 can open up at the same bond that closed the circle, yielding a linear intron (L-15). Reaction 4, the terminal G of L-15 can attack the bond between the first two U’s, yielding the circular intron C-19. Reaction 5, C-19 opens up to yield the linear intron L-19. A263 C262 G312 C311 U310 3′-OH GMP 5′-OH (a) (b) Figure 14.45 Two views of GMP held in a pocket of the 26S rRNA intron. (a) A cross-eyed stereogram that can be viewed in three dimensions by crossing the eyes until the two images merge. Carbon atoms of RNA, green; carbon atoms of G, yellow; phosphorus, lavender. Other atoms are standard colors. The GMP is at lower left. (b) Spacefilling model. Colors are as in part (a). (Source: Yarus, M., I. Illangesekare, S U M M A RY Nuclear mRNA precursors are spliced via a lariat-shaped, or branched, intermediate. In addition to the consensus sequences at the 59- and 39-ends of nuclear introns, and E. Christian, An axial binding site in the Tetrahymena precursor RNA. Journal of Molecular Biology. 222 (1991) f. 7c–d, p. 1005, by permission of Elsevier.) branchpoint consensus sequences also occur. In yeast, this sequence is nearly invariant: UACUAAC. In higher eukaryotes, the consensus sequence is more variable: YNCURAC. In all cases, the branched nucleotide is the final A in the sequence. The yeast branchpoint sequence also determines which downstream AG is the 39-splice site. wea25324_ch14_394-435.indd Page 432 432 13/12/10 7:24 AM user-f467 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile Chapter 14 / RNA Processing I: Splicing Splicing appears to take place on a particle called a spliceosome. Yeast and mammalian spliceosomes have sedimentation coefficients of about 40S and 60S, respectively. Genetic experiments have shown that base pairing between U1 snRNA and the 59-splice site of an mRNA precursor is necessary, but not sufficient, for splicing. The U6 snRNP also associates with the 59-end of the intron by base pairing. This association first occurs prior to formation of the lariat intermediate, but its character may change after this first step in splicing. The association between U6 and the splicing substrate is essential for the splicing process. U6 also associates with U2 during splicing. The U2 snRNA base-pairs with the conserved sequence at the splicing branchpoint. This base pairing is essential for splicing. U2 also forms vital base pairs with U6, forming a region called helix I, which apparently helps orient these snRNPs for splicing. The U4 snRNA base-pairs with U6, and its role seems to be to bind U6 until U6 is needed in the splicing reaction. The U5 snRNP associates with the last nucleotide in one exon and the first nucleotide of the next. This presumably lines the 59- and 39-splice sites up for splicing. The spliceosomal complex (substrate, U2, U5, and U6) poised for the second step in splicing can be drawn in the same way as a group II intron at the same stage of splicing. Thus, the spliceosomal snRNPs seem to substitute for elements at the center of catalytic activity of the group II introns, and probably have the spliceosome’s catalytic activity. Indeed, the catalytic center of the spliceosome appears to include Mg21 and a base-paired complex of three RNAs: U2 and U6 snRNAs, and the branchpoint region of the intron. Protein-free fragments of these three RNAs can catalyze a reaction related to the first splicing step. The spliceosome cycle includes the assembly, splicing activity, and disassembly of the spliceosome. Assembly begins with the binding of U1 to the splicing substrate to form a commitment complex. U2 is the next snRNP to join the complex, followed by the others. The binding of U2 requires ATP. U6 dissociates from U4, then displaces U1 at the 59-splice site. This ATP-dependent step activates the spliceosome and allows release of U1 and U4. The five snRNPs that participate in splicing all contain a common set of seven Sm proteins and several other proteins that are specific to each snRNP. The structure of U1 snRNP reveals that the Sm proteins form a doughnut-shaped structure to which the other proteins are attached. A minor class of introns with 59-splice sites and branchpoints can be spliced with the help of a minor spliceosome containing a variant class of snRNAs, including U11, U12, U4atac, and U6atac. The splicing factor Slu7 is required for correct 39-splice site selection. In its absence, splicing to the correct 39-splice site AG is specifically suppressed and splicing to aberrant AG’s within about 30 nt of the branchpoint is activated. U2AF is also required for 39-splice site recognition. The 65-kD U2AF subunit binds to the polypyrimidine tract upstream of the 39-splice site, and the 35-kD subunit binds to the 39-splice site AG. Commitment to splice at a given site is determined by an RNA-binding protein, which presumably binds to the splicing substrate and recruits other spliceosomal components, starting with U1. For example, the SR proteins SC35 and SF2/ASF commit splicing on human b-globin pre-mRNA and HIV tat pre-mRNA, respectively. In the yeast commitment complex, the branchpoint bridging protein (BBP) binds to a U1 snRNP protein at the 59-end of the intron, and to Mud2p near the 39-end of the intron. It also binds to the RNA near the 39-end of the intron. Thus, it bridges the intron and could play a role in defining the intron prior to splicing. The mammalian counterpart of BBP, SF1, may serve the a similar function, but in exon definition, in the mammalian commitment complex. The CTD of the Rpb1 subunit of RNA polymerase II stimulates splicing of substrates that use exon definition, but not those that use intron definition, to prepare the substrate for splicing. The CTD binds to splicing factors and could therefore assemble the factors at the ends of exons to set them off for splicing. The transcripts of many eukaryotic genes are subject to alternative splicing. This can have profound effects on the protein products of a gene. For example, it can make the difference between a secreted or a membrane-bound protein; it can even make the difference between activity and inactivity. In the fruit fly, the products of three genes in the sex determination pathway are subject to alternative splicing. Female-specific splicing of the tra transcript gives an active product that causes female-specific splicing of the dsx pre-mRNA, which produces a female fly. Male-specific splicing of the tra transcript gives an inactive product that allows default, or male-specific, splicing of the dsx premRNA, producing a male fly. Tra and its partner Tra-2 act in conjuction with one or more other SR proteins to commit splicing at the female-specific splice site on the dsx pre-mRNA. Such commitment is undoubtedly the basis of most, if not all, alternative splicing schemes. Alternative splicing is a very common phenomenon in higher eukaryotes. It represents a way to get more than one protein product out of the same gene, and a way to control gene expression in cells. Such control is exerted by splicing factors that bind to the splice sites and branchpoint, and also by proteins that interact with exonic splicing enhancers (ESEs), exonic splicing silencers (ESSs), and intronic silencing elements. SR proteins tend to bind to ESEs, while hnRNP proteins, such as hnRNP A1, bind to ESSs and intronic silencing elements. Group I introns, such as the one in the Tetrahymena 26S rRNA precursor, can be removed with no help from protein in vitro. The reaction begins with an attack by a wea25324_ch14_394-435.indd Page 433 13/12/10 7:24 AM user-f467 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile Review Questions guanine nucleotide on the 59-splice site, adding the G to the 59-end of the intron and releasing the first exon. In the second step, the first exon attacks the 39-splice site, ligating the two exons together and releasing the linear intron. The intron cyclizes twice, losing nucleotides each time, then linearizes for the last time. RNAs containing group II introns self-splice by a pathway that uses an A-branched lariat intermediate, just like the spliceosomal lariats. The secondary structures of the splicing complexes involving spliceosomal systems and group II introns are also strikingly similar. 433 15. Describe and show the results of an experiment that demonstrates that U5 contacts the 39-end of the upstream exon and the 59-end of the downstream exon during splicing. Make sure your experiment(s) provide positive identification of the RNA species involved, not just electrophoretic mobilities. 16. Describe and show the results of an experiment that demonstrates which bases in U5 can be cross-linked to bases in the pre-mRNA. 17. Summarize the evidence for a catalytic Mg21 in spliceosomal splicing. 18. Summarize the evidence that a mixture of spliceosonal RNA fragments can catalyze a reaction related to the first splicing step. REVIEW QUESTIONS 1. Describe and show the results of an R-looping experiment that demonstrates that an intron is transcribed. 2. Diagram the lariat mechanism of splicing. 3. Present gel electrophoretic data that suggest that the excised intron is circular, or lariat-shaped. 4. Present gel electrophoretic data that distinguish between a lariat-shaped splicing intermediate (the intron—exon-2 intermediate) and a lariat-shaped product (the excised intron). 5. The lariat model predicts an intermediate with a branched nucleotide. Describe and show the results of an experiment that confirms this prediction. 6. Describe and give the results of an experiment that shows that a sequence (UACUAAC) within a yeast intron is required for splicing. 7. Describe and show the results of an experiment that demonstrates that the UACUAAC sequence within a yeast intron dictates splicing to an AG downstream. 8. What role does the UACUAAC sequence play in the lariat model of splicing? 9. Describe and show the results of an experiment that demonstrates that yeast spliceosomes have a sedimentation coefficient of 40S. 10. Describe and show the results of an experiment that demonstrates that base pairing between U1 snRNA and the 59-splice site is required for splicing. 11. Describe and show the results of an experiment that demonstrates that base pairing between U1 and the 59-splice site is not sufficient for splicing. 12. What snRNP besides U1 and U5 must bind near the 59-splice site in order for splicing to occur? Present cross-linking data to support this conclusion. 13. Describe and show the results of an experiment that demonstrates that base pairing between U2 snRNA and the branchpoint sequence is required for splicing. In this experiment, why was it not possible to mutate the cell9s only copy of the U2 gene? 14. Besides base-pairing with the pre-mRNA, U6 base-pairs with two snRNAs. Which ones are they? 19. Draw a diagram of a pre-mRNA as it exists in a spliceosome just before the second step in splicing. Show the interactions with U2, U5, and U6 snRNPs. This scheme resembles the intermediate stage for splicing of what kind of self-splicing RNA? 20. Describe and show the results of an experiment that demonstrates that U1 is the first snRNP to bind to the splicing substrate. 21. Describe and show the results of an experiment that demonstrates that binding of all other snRNPs to the spliceosome depends on U1, and that binding of U2 requires ATP. 22. What are Sm proteins? 23. How do the characteristics of minor spliceosomes help show the importance of base-pairing between snRNAs and pre-mRNA sites? 24. Describe and show the results of an experiment that demonstrates that Slu7 is required for selection of the proper AG at the 39-splice site. 25. Describe a splicing commitment assay to screen for splicing factors involved in commitment. Show sample results. 26. Describe and give the results of a yeast two-hybrid assay that shows interaction between yeast branchpoint bridging protein (BBP) and two other proteins. What are the two other proteins, and where are they found with respect to the ends of the intron in the commitment complex? 27. Describe and give the results of an experiment that shows that the RNA polymerase II CTD stimulates splicing of pre-mRNAs that use exon definition. 28. Diagram the alternative splicing of the immunoglobulin m heavy-chain transcript. Focus on the exons that are involved in one or the other of the alternative pathways, rather than the ones that are involved in both. What difference in the protein products is caused by the two pathways of splicing? 29. Describe a computational and an experimental method to identify sequences that act as exonic splicing silencers (ESSs). 30. Describe and show the results of an experiment that demonstrates self-splicing by a group I intron. 31. Describe and show the results of an experiment that demonstrates that a guanine nucleotide is added to the end of a spliced-out group I intron. wea25324_ch14_394-435.indd Page 434 434 13/12/10 7:24 AM user-f467 /Volume/204/MHDQ268/wea25324_disk1of1/0073525324/wea25324_pagefile Chapter 14 / RNA Processing I: Splicing 32. Draw a diagram of the steps involved in autosplicing of an RNA containing a group I intron. You do not need to show cyclization of the intron. 33. Diagram the steps involved in forming the L-19 intron from the original excised linear intron product of the Tetrahymena 26S pre-rRNA. Do not go through the C-15 intermediate. A N A LY T I C A L Q U E S T I O N S 1. You are investigating a gene with one large intron and two short exons. Show the results of R-looping experiments performed with: a. mRNA and single-stranded DNA b. mRNA and double-stranded DNA c. mRNA precursor and single-stranded DNA d. mRNA precursor and double-stranded DNA 2. You have discovered a new class of introns that do not require any proteins for splicing, but do require several small RNAs. One of these small RNAs, V3, has a sequence of 7 nt (CCUUGAG) complementary to the 39-splice site. You suspect that base-pairing between V3 and the 39-splice site is required for splicing. Design an experiment to test this hypothesis and show sample positive results. 3. Diagram the mechanism of RNase T1 (or T2) action. Because this is the same mechanism used in base hydrolysis, how does this explain why DNA is not subject to base hydrolysis? 4. You are studying a grave human disease called b-thalassemia in which no b-globin protein is produced. You find that the b-globin gene’s coding region in people with this disease is normal, but the mRNA is over a hundred nucleotides longer than normal. You sequence the b-globin gene in these people and find a single base change within the gene’s first intron. Present a hypothesis to explain the absence of b-globin in these patients. 5. Consider the gene illustrated in Figure 14.38, but remove P2 and poly(A)1, so there is only one promoter (P1) and one polyadenylation site [poly(A)2]. How many different spliced mRNAs can now be produced by this gene? 6. Consider the RNA sequencing results in Figure 14.42b. Knowing the cutting specificities of each enzyme, how do we know (a) that the band at the bottom in the first lane represents G? (b) that the next band represents A? (c) that the eighth band from the bottom represents C? (d) that the 13th, 14th, and 15th bands from the bottom represent U’s? (Hint: PhyM cut inefficiently after U’s in this experiment.) 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