Who's on first? The U1 snRNP-5 splice site interaction and splicing

TIBS16-/VIAY1991

INTRONS, or intervening sequences, are present in the primary transcripts of many genes. Splicing is the biochemical process by which these introns are removed and the exons sewn together, to generate the mature RNA products. For pre-mRNA splicing, the chemical reaction takes place in two successive steps. First, cleavage of the 5' splice site occurs and the 5' phosphate of the first nucleotide of the intron forms a 2"5' phosphodiester bond with the 2'OH of the branchpoint nucleotide, creating a 'lariat'. Then 3' splice-site cleavage occurs concomitant with exon ligation. This second step forms the two final products of the splicing reaction, the ligated exons and the intron in lariat form. The pre-mRNA splicing pathway is similar, if not identical, to that fob lowed by group II self-splicing RNA molecules, suggesting that the premRNA pathway also occurs by two successive transesterification reactions ~. Well before this pathway was described and many of the transacting factors and small nuclear ribonucleoprotein particles (snRNPs) that participate in pre-mRNA splicing were identified, it was recognized that the 5' end of U1 snRNA is complementary to the 5' splice-site region consensus sequence (CAGGUAAGU) present in pre-mRNA introns. This insight led to the suggestion that U1 snRNP, the ribonucleoprotein particle that contains U1 snRNA, is involved in pre-mRNA splicingz3. The 5' splice site, the phosphodiester bond that sits between the last base of the 5' exon and the first base of the intron, lies within this pairing region. The consensus sequence of the tWO nucleotides that flank this bond is GG (the second G is the first base of the intron and is always (I) and the accepted view is that this sequence forms Watson-Crick base pairs with the completely conserved CC sequence at positions eight and nine of U1 snRNA (Fig. I). Subsequently, a number of studies have appeared that support the notion that the 5' end of U1 snRNA, and more generally U1 snRNP, is indeed important for pre-mRNA splicing. Early roamM. Roslmsh is at the Howard Hughes Medical Institute, Department of Biology, Brandeis University, Waltham, MA 02254, USA. B. S&aphln is at the European Molecular Biology Laboratory, Postfach 10.2209, Meyerhofstrasse 1, !)-6900 Heidelberg, FRG.

Who's on first The U1 s nRN 4::-splfce site.,: inte acti-oi{ and splicing

U1 small nuclear ribonucleoprotein (snRNP) is important for pre-mRNA splicing both in yeast (Saccharomyces cerevisiae) and mammalian systems. The RNA component of UI snRNP, U1 snRNA, interacts by base pairing with premRNA 5' splice sites. This article examines recent evidence suggesting that U1 snRNP is important for an early step in spliceosome assembly rather than a late step that contributes to the specificity of 5' splice-site cleavage.

malian experiments showed that U1 snRNP binds to 5' splice sites in vitro4. With the biochemical dissection of the splicing process, an oligonucleotidedirected RNase H inactivation strategy was used to show that an intact 5' end of U1 snRNA is required for in vitro splicings. In vivo experiments of Zhuang and Weiner demonstrated genetically that base pairing between the 5' end of U1 snRNA and the 5' splice junction contributes to splicing efficiency~. Subsequently, in vivo experiments in Saccharomyces cerevisiae gave rise to similar conclusions 7's. Mammalian studies designed to examine 5' splice-site

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choice (using 5' splice-site duplications or cryptic splice-site activation assays) showed that the sequence with the best complementarity to the 5' end of U1 snRNA is frequently selected as the 5' splice-site region (for examples see Refs

9-10. Given the importance of this interaction, two (non-exclusive) possibilities have been entertained for the biochemical contribution made by U1 snRNP to the splicing process. First, interaction of the U1 snRNP and the 5' splice-site region might occur at an early stage of the spliceosome assembly process, thereby contributing to 5' splice-site

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Rgure 1 ]he U1 snRNA-5' splice-site pairing interaction. In this schematic of the secondary structure of a typical mammalian U1 snRNA molecule, nucleotides 3--11 are sno~,,; ~,,,,.~;,ai::~1 to a consensus mammalian 5' splice-site region of a typical pre-mRNA. The nucleotides at the 5' end of U1 snRNA are show, irt bold letters, the first ten of which (in capital letters) are conserved between yeast and man (for example see Ref. 30). The large bold arrows indicate the usual positions of the 5' splice site (5'ss) and the 3' splice site (3'ss). The 3' splice site region (3'ss region) is depicted as encompassing the branchpoint region (BP). the polypyrimidine stretch (Py) and the last two nucleotides of the intron fAG).

© 1991,ElsevierSciencePublishersLtd,(UK) 0376-5067/91/$02.00

187

TIBS 1 6 - MAY1991

tribution to spliceosome assembly 12,~3, it was yeast in vitro studies that forcefully supported this hypothesis. As in the HeLa system "J3, a U2 snRNP-premRNA complex was the earliest stable substrate--snRNP complex that could be Is UI snRNPImportantfor early assemMy identified by non-denaturing gel electrophoresis in the first descriptions of events? Although previous experiments in the yeast spliceosome assembly pathHeLa cell extracts had provided hints way ~6.~7.Yet substrate challenge experthat UI snRNP might make an early con- iments indicated that pre-mRNA forms a stable complex (a 'commitment complex') committed to the splicing path3' exon exon 1 intron way in the absence of ATP and functional U2 snRNP (Ref. 18). Other in oilro 3' experiments, utilizing extracts from strains that expressed mutant UI snRNA genes, showed~that a proper UI UI, X snRNA-pre-mRNA pairing interaction is required for detectable U2 snRNP binding and splicing7. Independently, Ruby and Abelson TM used affinity chromatography assays to show that U1 snRNP binds to pre-mRNA in the absence of CC ATP and functional U2 snRNP; this binding precedes and is necessary for subsequent U2 snRNP binding. Although these latter experiments could not show that the U1 snRNP-pre-mRNA complex was a functional intermediate in splicing, they suggested that it might Ul be related to (or coincident with) the commitment complex defined in the former study 18. Both studies indicated a surprising role for the branchpoint ATP,U2 sequence in UI snRNP complex formation, i.e. prior to U2 snRNP addition. A more definitive and detailed picture emerged with the development of an in vivo strategy to prepare yeast splicing extracts depleted of individual snRNPs 2°. This genetic approach was highly effective and avoided potential complications associated with snRNP inactivation by oligonucleotide-directed RNase H cleavage. Analysis of spliceosome assembly in the extracts depleted SP U1 ? U2 of U2 snRNP, in combination with some modifications of existing gel electrophoresis methods, showed that a stable U1 snRNP-pre-mRNA substrate complex could be directly visualized on native gels. This complex was committed to proceed along the splicing pathway; pre~mRNAin the complex could be chased into spliceosomes as well as Rgure 2 Early events in spliceosome assembly. SP, spliceosomes (defined as U2 snRNP-containing into spliced intermediates and prodcomplexes); CC, commitment complexes; X, putative branchpoint interacting factor (or 3' ucts, confirming the biological validity splice-site region interacting factor) that also interacts with Ul snRNP (U1); 5'ss, 5' splice of the complex. These observations site; 3'ss, 3' splice site (i.e. the AG); bp, branchpoint region; Py, polypyrimidine stretch indicated that the U1 snRNP-containing region; U2, U2 snRNP. Although the polypyrimidine sequence is probably not very importcomplexes visualized on gels were ant for early complex formation in yeast (for examples see Refs 41,42), factor X is shown indistinguishable from the commitment interacting with both the branchpoint region and the polypyrimidine stretch to accommodate a role in mammalian spliceosome assembly for this putative factor. (Rgure modified complex that had been inferred from from Ref. 20.) indirect experiments. Consistent with 188 recognition, 5' splice-site choice and spliceosome assembly. Second, it might occur, or be significant, at a relatively late stage of the spliceosome assembly process, perhaps to stabilize an interaction that has already taken place or to contribute to 5' splice-site definition (the selection of the precise phosphodiester bond subjected to 'attack' by the branchpoint nucleotide). Without manipulating U1 snRNP itself, it is difficult

to define the precise contribution(s) that U1 snRNP, or the U1 snRNP-5' splice-site interaction, may make to splicing or to the spliceosome assembly process.

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TIBS16-MAY1991 the previous affinity chromatography experiments '9, it was also shown that there is a required order of snRNP binding to the substrate pre-mRNA; U1 snRNP binding occurred in the absence of ATP and U2 snRNP, whereas no U2 snRNP binding took place without U1 snRNP (Fig. 2). These experiments ~° also showed that recognition of the branchpoint region takes place in the absence of functional U2 snRNP, suggesting that a factor other than U2 snRNP mediates the initial recognition of the branchpoint region. This factor could be U1 snRNP itself. Alternatively, or in addition, U1 snRNP may interact with proteins that independently recognize the branchpoint region; this preferred interpretation has been included in the model by proposing the existence of a yeast factor (X in Fig. 2) that interacts with the branchpoint region prior to U2 snRNP addition. These experiments indicated a precise - and seemingly unambiguous order of events. They also provided strong evidence that the base-pairing interaction between U1 snRNA and the 5' splice site is important during these early stages of yeast spliceosome assembly. How does this pathway compare with the current view in mammalian systems? Although a definitive answer is lacking, some hints can be gleaned by comparing experiments where a similar or identical approach has been taken in the two systems. Barabino et al. 2~ have recently described experiments that exploit the elegant antisense affinity chromatography procedures developed by the EMBL groups. Like the yeast genetic approach referred to above 2°, this biochemical procedure effects a. dramatic and specific depletion of U1 snRNP from HeLa cell splicing extracts. Characterization of the depleted extracts showed that U1 snRNP is required for stable U2 snRNP binding to the branchpoint region. As the binding of U2 snRNP is an early event in spliceosome assembly, these data indicate that U1 snRNP is indeed critical at a relatively early stage of the mammalian spliceosome assembly process. These workers also explored the nature of this U1 snRNP requirement for U2 snRNP binding in some detail. Previous results had indicated that stable U2 snRNP binding often has no demonstrable requirement fo: a 5' splice }unction (for example see Refs 14,22,23). In these experiments

as well21, deletion of the 5' splicesite region or oligonucleotide-directed masking of the 5' end of U1 snRNA had no effect on U2 snRNP binding to the pre-mRNA substrate. Yet U2 snRNP binding retained its U1 snRNP dependence, even with a deletion substrate lacking any obvious 5' splice junction 21. The most likely interpretation of these mammalian experiments is that the U1 snRNP potentiates U2 snRNP binding without detectable formation of the accepted U1 snRNA-5' splice site basepairing interaction. These results also suggest that, at least in mammalian systems, UI snRNP might act to promote U2 snRNP-premRNA binding without prior formation of a stable U1 snRNP-pre-mRNA complex. Yet there is recent evidence for the formation of a U1 snRNP-pre-mRNA complex in mammalian extracts 24. Perhaps U1 snRNP interacts strongly with the pre-mRNA at a location other than the 5' splice-site region, so that the rate and extent of U2 snRNP binding is insensitive to the absence of the basepairing interaction. There are indications that U1 snRNP interacts with the branchpoint-polypydmidine--3' spli,~site region (for examples see Ref. 12; J. G. Patton and B. Nadal-Ginard, pets. commun.). As there are at least two mammalian proteins that interact with the branchpoint-polypyrimidine--3' splicesite region~'27, one or more of them may bridge the early interaction of U1 snRNP with this region (like the yeast factor X in Fig. 2, but with a stronger interaction with the polypyrimidine stretch than with the branchpoint region). In this view, the yeast-mammalian differences are largely quantitative, with a more important mammalian U1 snR_NP-X interaction and a less important mammalian UI snRNP-5' splice-site region interaction relative to the interactions in yeast. Of course, quantitative differences in crude extracts may have little biological significance, due, for example, to the relative ability of some splicing factors to survive extract preparation. There are of course other interpretations for the apparent differences in the early assembly events. In mammals, the U1 snRNP-5' splice-site region interaction might follow U2 snRNP binding, implying that, at least in vitro, some aspects of 5' splice-site recognition or definition might not be so 'early' and might follow 3' splice-site recognition or definition. The U1 snRNP-5' splice-site interaction might even be optional

under some circumstances in mammalian cells or extracts (see below). These considerations emphasize the possibility that there are qualitative differences between yeast and mamma]~ in the order or the interdependence of certain early steps in spliceosome assembly, reflecting perhaps the substantial difference between yeast and mammals in U1 snRNA size and structure ~a0.

Is UlsnRNP important for 'late' events in ~dbl~ or ~ M ~ A different but equally contemporary perspective emphasizes a relatively late role for U1 snRNP during mammalian spliceosome assembly. This view is based largely on in vitro experiments that examined the effects of 5' splicesite mutations that changed the highly conserved GU to UU or CU in the rabbit [3-globin intron31. Rather than activating cryptic splice sites, these mutations led to a shift of the 5' splice site one nucleotide upstream of the normal 5' splice site. Because splicing occurred at the location that showed the best complementarity to the 5' end of U1 snRNA, the authors proposed that 5' splice-site cleavage occurs within the paired region opposite the two conserved C residues of U1 snRNA (Fig. I). However, experiments in yeast appear inconsistent with this rule. Mutants at position five of the intron gave rise to aberrant cleavage events a few nucleotides upstream or downstream of the normal 5' splice site 32~. These new cleavage sites showed no obvious sequence homology to normal 5' splice sites. Also, products of these aberrant events were unable to proceed through the second step of splicing, i.e. the two reaction intermediates, 5' exons and lariat intermediates, accumulated. To examine the role of U1 snRNA-premRNA base-pairing in 5' splice-site selection, U1 snRNAs with compensating mutations were constructed and introduced into yeast. Although these mutant snRNAs were functional, they could not correct the cleavage defects of the mutant pre-mRNAs7's. Two recent yeast studies have reinforced and extended these observations 3s.~. The addition of exon mutations to an aberrant cleavage site transformed it into a functional 5' splice site, both in vivo and in vitro~. But the location of the U1 snRNA-pre-mRNA interaction was not affected by the exon mutations, and 5' splice-site selection was still independent of this pair189

TIBS 1 6 - M A Y1991

ing. These experiments were therefore unable to confirm the contribution of the standard U1 snRNA-prc-r~RNApairing to the selection of the precise 5' splice site. The other study suggested that U5 snRNA is involved in defining the specificity of 5' splice-site cleavage~. As the aberrant 5' splice sites (in one case 12 nucleotides from the proper 5' splice site) required the presence of the original 5' splice site, it is possible that the aberrant events were dependent upon a U1 snRNP-5' splice-site .i~teract!o~,.at the usual location. Both studies suggest that yeast U1 snRNP may contribute only to the early stages of spliceosome assembly that identify the 5' splice-site region and potentiate U2 snRNP binding rather than to the later stages of assembly that impact more directly on the efficiency and specificity of the cleavage and ligation events. The situation may be the same in mammals, or yeast and mammals might differ in the way that 5' splice-site specificity is determined. The possible involvement of other factors in 5' splice-site selection is reinforced by a consideration of trypanosome trans-splicing. U1 snRNP and U1 snRNA have not been identified in these organisms, yet 5' splice sites within the spliced leader (SL) RNA resemble those of yeast and mammals37-~9.it has also been suggested that the SL RNA contains its own UI snRNP.like activity 37. Very recent experiments indicate that some chimeric substrates containing a trypanosomatid 5' SL sequence, or derivatives thereof, are accurately spliced in HeLa cell extracts in the absence of functional UI snRNP (Ref. 40). If there are other factors that interact with 5' splice sites during the

later stages of spliceosome assembly, they might contribute to 5' splice-site cleavage both in trypanosomes and in the HeLa cell extract-chimeric substrate experiments. The mystery could then be rephrased: how does the SL, and/or some other aspect of the chimeric substrates, bypass or reduce the normal requirement for functional U1 snRNP in the recognition or identification of the 5' splice-site region during the early phases of spliceosome assem-

bly? From this perspective, the combi, nation -.f SL-containing substrates and

mammalian extracts without U1 snRNP

activity should continue to provide insights not only about trans-splicing but also about the role of U1 snRNP in

mammalian splicing. References 1 Jacquier,~, (199o) Trends Biochem. Sci. 15, 351-354 2 Lerner, M. R., Boyle,J. A., Mount, S. M., Wolin, S. and Steitz, J. A. (1980) Nature 283, 220-224 3 Rogers,J. and Wall, R. (1980) Proc. Nat/Acad. Sci. USA 77, 1877-1879 4 Mount, S. M., Pettersson, I., Hinterberger, M., Karmas, A. and Steitz, J. A. (1983) Cell33, 509-518 5 Kramer,A., Keller,W., Appel, B. and Luhrmann, R. (1984) Cell38, 299-307 6 Zhuang, Y. and Weiner,A. M. (1986) Ce/146, 827-835 7 S~raphin, B., Kretzner, L. and Rosbash, M. (1988) EMBO J. 7, 2533-2538 8 Siliciano, P. G. and Guthrie, C. (1988) Genes Dev. 2, 1258-1267 9 Eperon, L. P., Estibeiro,J. P. and Eperon, I. C. (1986) Nature 324, 280-282 10 Nelson, K. K, and Green, M. R. (1988) Genes Dev. 2, 319-329 11 Nelson, K. K. and Green, M. R. (1990) Proc. Natl Acad. Sci. USA 87, 6253-6257 12 Zillmann, M., Rose, S. D. and Berget, S. M. (1987) Mol. Cell, Biol. 7, 2877-2883 13 Kramer,A. (1988) Genes Dev. 2, 1155-1167 14 Konarska, M. M. and Sharp, R A. (1986) Cell 46, 845-855 15 Lamond, A. I., Konarska, M. M., Grabowski, P. J.

and Sharp, P. A. (1988) Proc. Natl Acad. ScL USA 85, 411-415 ze Pikielny, C. W., Rymond, B. C. and Rosbash, M. (1986) Nature 324, 341-345

17 Cheng, S. C. and Abelson, J. (1987) Genes Dev.

1,1014-1027 Legrain, P., S6raphin, B. and Rosbash, M. 18 (1988) MoL Cell. Biol. 8, 3755-3760 19 Ruby,S. W. and Abelson, J. (1988) Science 242, 1028-1035 20 S6raphin, B. and Rosbash,M. (1989) Cell 59, 349-358 21 Barabino, S. M. L., Blencowe, 8. J., Ryder, U., Sproat, B. S. and Lamond,A. I. (1990) Cell63, 293-302 22 Frendewey,D. and Keller,W. (1985) Cell42, 355-367 23 Bindereif, A. and Green, M. R. (1987) EMBO J. 6, 2415-2424 24 Reed, R. (1990) Proc. Natl Acad. Sci. USA 87, 8031-8035 25 Ruskin, B., Zarnore, P. D. and Green, M. R. (1988) Cell 52, 207-219 26 Zamore, P. D. and Green, M. R. (1989) Proc. Natl Acad. Sci. USA 86, 9243-9247 27 Garcia-Blanco,M. A., Jamison, S. F. and Sharp, R A. (1989) Genes Dev. 3, 1874-1886 28 Kretzner, L., Rymond, B. C. and Rosbash, M. (1987) Cell 50, 593-602 29 Siliciano, P. G., Jones, M. H. and Guthrie, C. (1987) Science 237, 1484-1487 30 Kretzner, L., Krol, A. and Rosbash, M. (1990) Proc. Natl Acad. ScL USA 87, 851-855 31 Aebi, M., Homig, H. and Weissmann, C. (1987) Ce/150, 237-246 32 Parker, R. and Guthrie, C. (1985) Cell41, 107-118 33 Jacquier, A., Rodriguez,J. R. and Rosbash, M. (1985) Cell43, 423-430 34 Fouser,L. A. and Friesen,J. D. (1986) Cell45, 81-93 35 S(~raphin,B. and Rosbash, M. (1990) Cell63, 619-629 36 Newman,A. and Norman,C. (in press) 37 Bruzik, J, P., Van Doren, K., Hirsh, D. and Steitz, J. A. (1988) Nature 335, 559-562 38 Mottram,J., Perry, K, L., Uzardl, R M., Luhrmann, R., Agabian, N. and Nelson, R. G. (1989) Mol, Cell. Biol. 9, 1212-1223 39 Agabian, N. (1990) Cell61, 1157-1160 40 8ruzik, J, R and Steitz, J. A. (1990) Cell62, 889-899 41 Rymond,B. C. and Rosbash, M. (1985) Nature 317, 735-737 42 Rymond,B. C., Torrey,D. D. and Rosbash, M. 0 987) Genes Dev. 1, 238-246

Do you enjoy teasing your brain to untangle the TIBS acrostics? Naomi Lipsky, who compiles these puzzles for us, has issued a challenge to TIBS readers to devise their own puzzle. Naomi will judge this competition and we will publish the winning entry in the August issue of TIBS. The winner will receive a one-year subscription to TIBS. You should choose a quotation of 190-240 letters. This should be fitted to a 'crossword' grid, with the squares numbered sequentially. There should be 24-29 clues, each of 7-9 letters. Label each clue with a letter of the alphabet and assign to each letter of the solutions the number from the corresponding square on the grid. Likewise, transfer the letters identifying the clues to the grid squares. The initial let. ters of the solutions should form an acrostic spelling o,,, ,'he =uthor's name and title of the work from which the quotation is taken. The source must be an original work, although tram,|~,~,ons are acceptable. A quotation in a biography or review should not be used. The title of either the chapter or book should be incorporated ~, he puzzle. Please supply original title, English title, publisher, translator or editor, year published and page. The author should be a reputable, practising scientist, not a science historian or a biographer. The author's full last name should be included, The quotation should be relevant to the TIBS reader and may be edited for brevity, but the whole should make sense and convey the author's intent. Humorous or morally uplifting passages are preferred. Please supply the entire original quotation, as well as the edited version. Clues should be brief and direct, and may include numbers and symbols. Archaic, slang, proper names and abbreviated words are fine, as long as they are intemationally accepted. All words must be technical or have a technical definition. Please supply a reference for every clue used, i.e. a technical publication or dictionary where the word appears. Entries should be received by 1 June 1991. For solution to Lipsky acrostic (December) see p. 186. 190