Three Novel Functional Variants of Human U5 Small Nuclear RNA

Vol. 12, No. 2

MOLECULAR AND CELLULAR BIOLOGY, Feb. 1992, p. 734-746

0270-7306/92/020734-13$02.00/0

Three Novel Functional Variants of Human U5 Small Nuclear RNA ERIK J. SONTHEIMER AND JOAN A. STEITZ*

Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Avenue, New Haven, Connecticut 06536-0182 Received 25 September 1991/Accepted 18 November 1991

We have identified and characterized three new variants of U5 small nuclear RNA (snRNA) from HeLa cells, called U5D, U5E, and U5F. Each variant has a 2,2,7-trimethylguanosine cap and is packaged into an Sm-precipitable small nuclear ribonucleoprotein (snRNP) particle. All retain the evolutionarily invariant 9-base loop at the top of stem 1; however, numerous base changes relative to the abundant forms of U5 snRNA are present in other regions of the RNAs, including a loop that is part of the yeast U5 minimal domain required for viability and has been shown to bind a protein in HeLa extracts. USE and U5F each constitute 7% of the total U5 population in HeLa cells and are slightly longer than the previously characterized human U5 (A, B, and C) species. USD, which composes 5% of HeLa cell U5 snRNAs, is present in two forms: a full-length species, USDL, and a shorter species, USDS, which is truncated by 15 nucleotides at its 3' end and therefore resembles the short form of U5 (snR7S) in Saccharomyces cerevisiae. We have established conditions that allow specific detection of the individual U5 variants by either Northern blotting (RNA blotting) or primer extension; likewise, USE and USF can be specifically and completely degraded in splicing extracts by oligonucleotidedirected RNase H cleavage. All variant U5 snRNAs are assembled into functional particles, as indicated by their immunoprecipitability with anti-(U5) RNP antibodies, their incorporation into the U4/US/U6 tri-snRNP complex, and their presence in affinity-purified spliceosomes. The higher abundance of these U5 variants in 293 cells compared with that in HeLa cells suggests possible roles in alternative splicing.

U small nuclear ribonucleoproteins (U snRNPs) are complexes of proteins and small RNAs (U RNAs) found in nuclei of all eukaryotic cells examined (reviewed in reference 6). In mammalian cells, some U snRNPs are of high abundance (Ul through U6 are present in 105 to 106 copies per cell), while others (U7 through U13) are of lower abundance (103 to 104 per cell). Mammalian U small nuclear RNAs (snRNAs) share several common features: they are small (U5DS 1

2

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FIG. 5. Northern blot analysis of RNAs present in affinitypurified spliceosomes. Splicing complexes were formed on an adenovirus pre-mRNA substrate in 293 cell nuclear extract, fractionated, and affinity purified as described in Materials and Methods. The resulting RNAs were fractionated on a 10% denaturing polyacrylamide gel, blotted, and probed for the Ul, U2, U4, U5, and U6 snRNAs, as indicated on the right. Lane 1, pBR322 MspI DNA markers (sizes in nucleotides are shown on the left); lane 2, anti-Sm-immunoprecipitated RNAs from 293 cells, used as markers; lane 3, RNAs from a complete reaction; lane 4, RNAs from a control reaction in which the ATP and creatine phosphate were omitted; lane 5, RNAs from a control reaction in which the substrate pre-mRNA was omitted.

tions containing a biotinylated adenoviral pre-mRNA in a 10 to 30% glycerol gradient, followed by affinity selection of spliceosomal peak fractions with stretavidin-agarose (21). Two control reactions were performed in parallel, omitting either pre-mRNA or ATP and creatine phosphate. Spliceosomal peaks were identified as described in Materials and Methods, and affinity-selected material was analyzed by Northern blotting of a high-resolution gel with antisense Ul, U2, U4, U5, and U6 RNAs as probes. Results are shown in Fig. 5. Low levels of all five spliceosomal snRNAs are present in the control samples (lanes 4 and 5), probably because of nonspecific interactions between snRNPs and the straptavidin-agarose beads. The RNA sample from the complete reaction, however, is greatly enriched for the U2, U4, U5, and U6 snRNAs, demonstrating their presence in the spliceosome. Ul snRNA levels are not enhanced in the complete reaction; it was previously observed that the Ul snRNP-pre-mRNA interaction is sensitive to heparin (5, 21). It is clear that all three of the new U5 variants (including the truncated form of USD) are enriched in the complete spliceosome (lane 3); although it is possible that the spliceo-

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somes containing the variant U5 snRNAs do not actually support splicing, we favor the interpretation that each of these snRNAs assembles into functional splicing complexes. Variant U5 snRNPs are immunoprecipitated with antibodies against U5-specific proteins. Since U5D, U5E, and U5F each have sequence variations in a loop which binds protein (see Discussion), we considered the possibility that these snRNPs have an altered protein composition relative to the abundant forms of U5. Immunoprecipitation with antibodies directed against proteins specific to the U5 particle was used to examine the presence or absence of those proteins in the variant U5 snRNPs. An antiserum (LaJ) that specifically immunoprecipitates the U5 snRNP and recognizes a single 52-kDa protein on immunoblots has recently been identified from a patient with an autoimmune disease (42a); purified U5 snRNPs from HeLa cells have previously been shown to contain a 52-kDa protein (2). We used LaJ to immunoprecipitate 293 cell nuclear extracts and then assessed the presence of the variant U5 snRNAs by Northern blotting. Figure 6A shows that all of the U5 species are quantitatively immunoprecipitated, even when the NaCl concentration in the wash buffer is raised from 100 to 500 mM (lanes 6 to 10). (The small amounts of Ul, U4, and U6 snRNAs that remain bound even at high salt concentrations are due to a very low background of anti-Sm specificity in the LaJ serum [42a].) Strikingly, the U4 and U6 snRNAs are coprecipitated at low salt concentrations, most likely through the formation of the U41U51U6 complex; coprecipitation decreases as the salt concentration is raised, consistent with previous reports that a high salt concentration disrupts the tri-snRNP particle (4). A monoclonal mouse IgM antibody (H386) originally raised against the Ul 70K protein (48) has been found to cross-react fortuitously with a 100-kDa protein unique to the U5 snRNP (4). U5 and U41U5/U6 peak fractions from a glycerol gradient identical to that shown in Fig. 4 were used as the starting material for immunoprecipitations with this antibody. (Fractions 6 and 7 were used as the U4/U5/U6 sample, while fractions 10 and 11 were used as the U5 peak since they contain significant amounts of the U5 snRNP and are well separated from the U41U5/U6 tri-snRNP.) In the top panel of Fig. 6B, immunoprecipitated RNAs were labeled with 5'-32P-pCp and T4 RNA ligase and subjected to electrophoresis in a 9% polyacrylamide denaturing gel. LaJ anti-(U5) RNP patient serum, used as a positive control, efficiently precipitates U5 (lane 1) and U41U5/U6 (lane 4) particles. (The fastest-migrating band in the upper panel of lane 1 is primarily USDS, not U6 [data not shown]; the detection of some U4 snRNA in lane 1 is due to the presence of a small amount of the U4/U5/U6 complex in the U5 peak fraction.) When a goat anti-mouse IgM secondary antibody (required to bind H386 to protein A-Sepharose) was used alone as a negative control, only background levels of snRNAs were detected from either the U5 or U41U5/U6 sample (lanes 2 and 5); when the H386 antibody was included, specific immunoprecipitation of the Ul and U5 snRNPs is observed (lanes 3 and 6). In addition, the U4 and U6 snRNAs are coprecipitated from the U4/U5/U6 fraction (lane 6; U6 appears to be underrepresented, since it is very inefficiently labeled with pCp and T4 RNA ligase [e.g., compare the relative labeling of U4, U5, and U6 in Fig. 1A, which does not reflect their equivalent abundance in anti-Sm precipitates]). When RNA samples from the same experiment were fractionated to resolve the U5 population, blotted, and probed for U5 (bottom panel), specific immunoprecipitation of each of the variant forms was observed.

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These experiments indicate that at least two proteins of 52 and 100 kDa unique to the U5 snRNP are present in the variant U5 snRNPs. However, the possibility of an altered distribution of any of the five to six other U5-specific proteins cannot be excluded. Oligonucleotide-directed RNase H degradation of U5 variants. It was previously reported that loop A of U5 snRNA is accessible to oligodeoxynucleotide-directed RNase H cleavage, although approximately 20% of U5 snRNA from HeLa cells remained uncleaved by this treatment (8). Since USD, USE, and USF together compose about 20% of the HeLa U5 population and have different sequences in loop A, we considered the likelihood that the residual uncleaved molecules might represent these variant U5 snRNAs.

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We incubated HeLa nuclear extracts with variant-specific oligonucleotides and RNase H in the presence of ATP, phenol extracted the RNA, divided the sample into fifths, and assessed the survival of each U5 snRNA by reverse transcription with the appropriate variant-specific primer (Fig. 7). A U2-specific primer was included in each reaction as a positive control. The USF primer extension products are visible only in the darker exposure in the bottom panel. The control reaction incubated without oligonucleotide (lanes 1 to 5) indicates that all U5 species are present. Likewise, incubation with a U2-specific oligonucleotide (lanes 6 to 10) results in cleavage of U2 snRNA but no cleavage of any of the forms of U5. When an oligonucleotide complementary to the loop A sequences of U5A and USB was used, the levels of these abundant forms were diminished by more than 95% while the levels of the variants were unchanged (lanes 11 to 15). Lanes 16 to 20 (bottom panel) and 26 to 30 reveal that both USE and USF can be selectively and completely (>95%) degraded by incubation with variant-specific complementary oligonucleotides. From lanes 21 to 25 it is clear that although the levels of U5D are diminished following RNase H targeting, depletion is far from complete. USD is extremely uridine rich in its loop A region (including a stretch of 8 of 12 bases); it is possible that the extreme instability of deoxyadenosine-ribouridine base pairs (11, 37) renders the U5D-specific oligonucleotide ineffective in directing RNase H degradation. Similar results were obtained

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DISCUSSION The identification of three novel variants of U5 snRNA (U5D, USE, and U5F) brings the number of completely sequenced HeLa cell U5 snRNAs to five. Previously, complete sequences of U5A and USB1 and 3'-end sequences of five additional HeLa U5 variants (USB2-4 and USC1-2) were reported (9, 29). Thus, the U5 population is by far the most varied of the U snRNAs in mammalian cells. The existence of U5D in both truncated and full-length forms in HeLa cells is analogous to the situation in the yeast S. cerevisiae, in which the snR7 transcript (yeast U5) exists in two alternative forms that differ in the presence or absence of the 3'-terminal stem-loop (44). Both snR7 transcripts are products of the same gene, and it is not known whether the truncated form arises from nucleolytic degradation (processing?) of a fulllength transcript or from transcription termination at two sites. Both short and long forms are found in the yeast spliceosome (49). The fact that numerous U5 RNA species exist in HeLa cells raises a number of intriguing questions. First of all, how are so many variant genes maintained? Ul and U2 snRNAs

encoded by multigene families in human cells, but the homogeneity of snRNA sequences within each family appears to be actively preserved (reviewed in reference 13). By what mechanism has the human U5 gene family managed to escape the action of such homogenizing mechanisms? Also, does the fact that the various U5 forms are expressed at different levels in HeLa cells reflect differences in gene copy number, promoter strength, RNA stability or transport, or some combination of these? Does the pattern of expression of U5 variants differ in a tissue- or developmental stagespecific manner? Such effects have been observed for U RNAs in a range of organisms. Specific examples include the embryonic and adult forms of Ul genes in mice (36) and Ul and U4 Xenopus genes (18, 34, 35), the different ratios of chicken U4B and U4X snRNAs in various tissues and developmental stages (27), and the variation in expression patterns of some U snRNAs during plant development and differentiation (22). Second, can the identification of these novel U5 forms aid in understanding the role played by the U5 snRNP in the splicing process? Biochemical analysis of U5 snRNP function in mammalian in vitro splicing systems has been hampered by the inability to specifically and completely degrade the U5 snRNP by oligonucleotide-directed RNase H digesare

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tion. Previous attempts yielded approximately 80% cleavage when loop A of USA and U5B1 was targeted (8). We have tried to achieve complete destruction of the US population in in vitro splicing extracts from HeLa cells by using a mixture of oligonucleotides directed against the loop A sequences of all of the different forms of US, but we still have not been able to specifically inhibit pre-mRNA splicing; the levels of oligonucleotide required to achieve total US degradation were so high that equivalent amounts of nonspecific oligonucleotides inhibited splicing to the same extent (data not shown). Two other approaches to U5 snRNP depletion in splicing extracts (affinity selection with modified 2'-Omethyl oligoribonucleotides [30] and immunochemical-biochemical fractionation [57]) have indicated that the U5 snRNP is required for spliceosome assembly and both steps of the splicing reaction. Perhaps the most important question raised by our observations is whether the differences in sequence between the variant U5 snRNAs underlie subtle differences in function. For instance, might these variants participate differentially in alternative splicing events? The sequence differences in loop B are particularly interesting in this regard. This loop lies within the minimal domain of the yeast U5 (snR7) required for viability; moreover, a gene for human U5A snRNA cannot complement an snr7 null allele unless its loop B sequence has been replaced by that of yeast (18a). (It is interesting to note that the loop B sequence of U5F more closely matches that of snR7; therefore, human U5F might succeed in complementation where U5A did not.) Furthermore, the protein composition of the U5 snRNP in HeLa cells is unusually complex (2), and chemical modification experiments indicate that loop B is bound by protein in splicing extracts (8). Since protein binding specificity can be altered by very subtle differences in single-stranded RNA loop sequences (3, 25, 50), it is possible that the protein composition differs among some or all of the variants. Any such difference could mediate an altered function. Circumstantial evidence suggests that variant U5 snRNAs may be involved in alternative splicing of the simian virus 40 early transcript. This pre-mRNA has a single 3' splice site which can be joined to either of two 5' splice sites; use of the proximal 5' splice site gives rise to mRNA for the small t antigen, while use of the distal site yields large T mRNA. HeLa and 293 cells produce different ratios of the two spliced mRNAs, with large T mRNA predominating in HeLa and small t mRNA predominating in 293 cells (19). The same specificity can be observed in vitro with splicing extracts made from the two cell types (20). Analysis of affinitypurified splicing complexes formed on a biotinylated premRNA following incubation in nuclear extracts from the two cell types showed that a longer US variant with a 3'-end sequence very similar to that of USF was present at higher levels in the 293 extracts than in the HeLa extracts (19a). We have confirmed the observation that USF is more abundant in 293 cells than in HeLa cells and have also shown that the same holds true for U5DS and U5E (data not shown). It is conceivable that a variant U5 snRNA facilitates splicing of the unusually short (66 nucleotides) small t intron. Subsequently, a protein purified from 293 extracts, designated ASF, was shown to dramatically enhance small t splicing when added to HeLa extracts (20); it is possible that this factor somehow modulates the relative usage of different forms of U5 in the splicing process. Since USE and USF can now be specifically and completely degraded by oligonucleotide-directed RNase H digestion, the possibility that they

MOL. CELL. BIOL.

alter 5' splice site choice during splicing of simian virus 40 early pre-mRNA in vitro can be directly tested. The possibility that the U5 snRNP can affect 5' splice site utilization has been dramatically reinforced by the recent results of Newman and Norman (41), who demonstrated that a set of mutations in loop C of yeast U5 can activate nearby cryptic 5' splice sites when the G in position 1 of an intron is changed to an A. (The loop C sequence is invariant among eukaryotic U5 snRNAs [9, 44].) Furthermore, one of these U5 mutations allows use of the natural 5' splice site in spite of the G1-to-A intron mutation. Although none of the U5 variants in HeLa cells are altered in the conserved loop C sequence, these results indicate that the U5 snRNP can play a role in 5' splice site selection and/or maintenance of 5' splice site fidelity. They also strengthen the possibility that these variants modulate 5' splice site choice in alternatively spliced pre-mRNAs such as the simian virus 40 early transcript. ACKNOWLEDGMENTS We acknowledge Yutaka Okano for generously providing the LaJ anti-(U5) RNP patient serum, as well as for helpful discussions and communication of results prior to publication. We thank Reinhard Luhrmann and Adrian Krainer for providing the H386 and the K121 monoclonal antibodies, respectively; Mary Schuler (Urbana), Dan Frank and Chris Guthrie (UCSF), and Hui Ge and Jim Manley (Columbia) for sharing data prior to publication; and Alan Weiner for comments on the manuscript. We also thank the entire Steitz lab (particularly David Toczyski, Kazio Tyc, David Wassarman, and Karen Wassarman) for advice and discussions and Susanna Lee, David Toczyski, David Wassarman, and Karen Wassarman for critical reading of the manuscript. This research was supported by grant GM26154 from the National Institutes of Health. REFERENCES 1. Anderson, G. J., M. Bach, R. Luhrmann, and J. Beggs. 1989. Conservation between yeast and man of a protein associated with U5 small nuclear ribonucleoprotein. Nature (London) 342:819-821. 2. Bach, M., G. Winkelmann, and R. Luhrmann. 1989. 20S small nuclear ribonucleoprotein U5 shows a surprisingly complex protein composition. Proc. Natl. Acad. Sci. USA 86:6038-6042. 3. Baserga, S. J., X. W. Yang, and J. A. Steitz. 1991. An intact box C sequence in the U3 snRNA is required for binding of fibrillarin, the protein common to the major family of nucleolar snRNPs. EMBO J. 10:2645-2651. 4. Behrens, S.-E., and R. Luhrmann. 1991. Immunoaffinity purification of a [U4/U6.U5] tri-snRNP from human cells. Genes Dev. 5:1439-1452. 5. Bindereif, A., and M. R. Green. 1987. An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly. EMBO J. 6:2415-2424. 6. Birnstiel, M. L. (ed). 1988. Structure and function of major and minor small nuclear ribonucleoprotein particles. Springer-Verlag KG, Berlin. 7. Black, D. L., B. Chabot, and J. A. Steitz. 1985. U2 as well as Ul small nuclear ribonucleoproteins are involved in premessenger RNA splicing. Cell 42:737-750. 8. Black, D. L., and A. L. Pinto. 1989. U5 small nuclear ribonucleoprotein: RNA structure analysis and ATP-dependent interaction with U4/U6. Mol. Cell. Biol. 9:3350-3359. 9. Branlant, C., A. Krol, E. Lazar, B. Haendler, M. Jacob, L. Galego-Dias, and C. Pousada. 1983. High evolutionary conservation of the secondary structure and of certain nucleotide sequences of U5 RNA. Nucleic Acids Res. 11:8359-8367. 10. Brow, D., and C. Guthrie. 1988. Spliceosomal RNA U6 is remarkably conserved from yeast to mammals. Nature (London) 334:213-218. 11. Buvoli, M., G. Biamonti, S. Riva, and C. Morandi. 1987.

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Hybridization of oligodeoxynucleotide probes to RNA molecules: specificity and stability of duplexes. Nucleic Acids Res. 15:9091. 12. Chabot, B., D. L. Black, D. M. LeMaster, and J. A. Steitz. 1985. The 3' splice site of pre-messenger RNA is recognized by a small nuclear ribonucleoprotein. Science 230:1344-1349. 13. Dahlberg, J. E., and E. Lund. 1988. The genes and transcription of the major small nuclear RNAs, p. 38-70. In M. L. Birnstiel (ed.), Structure and function of the major and minor small nuclear ribonucleoprotein particles. Springer-Verlag KG, Berlin. 14. Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11:1475-1489. 15. Donis-KeUler, H. 1980. PhyM: an RNase activity specific for U and A residues useful in RNA sequence analysis. Nucleic Acids Res. 8:3133-3142. 16. Donis-Keller, H., A. M. Maxam, and W. Gilbert. 1977. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 4:2527-2538. 17. England, T. E., A. G. Bruce, and 0. C. Uhlenbeck. 1980. Specific labeling of 3' termini of RNA with T4 RNA ligase. Methods Enzymol. 65:65-74. 18. Forbes, D. J., M. W. Kirschner, D. Caput, J. E. Dahlberg, and E. Lund. 1984. Differential expression of multiple Ul small nuclear RNAs in oocytes and embryos of Xenopus laevis. Cell 38:681-689. 18a.Frank, D., and C. Guthrie. Personal communication. 19. Fu, X.-Y., and J. L. Manley. 1987. Factors influencing alternative splice site utilization in vivo. Mol. Cell. Biol. 7:738-748. 19a.Ge, H., and J. Manley. Personal communication. 20. Ge, H., and J. L. Manley. 1990. A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro. Cell 62:25-34. 21. Grabowski, P. J., and P. A. Sharp. 1986. Affinity chromatography of splicing complexes: U2, U5, and U4+U6 small nuclear ribonucleoprotein particles in the spliceosome. Science 233: 1294-1299. 22. Hanley, B. A., and M. A. Schuler. Nucleic Acids Res., in press. 23. Heintz, N., and R. G. Roeder. 1984. Transcription of human histone genes in extracts from synchronized HeLa cells. Proc. Natl. Acad. Sci. USA 81:2713-2717. 24. Jamieson, D. J., B. Rahe, J. Pringle, and J. D. Beggs. 1991. A suppressor of a yeast splicing mutation (prp8-1) encodes a putative ATP-dependent RNA helicase. Nature (London) 349: 715-717. 25. Jones, M. H., and C. Guthrie. 1990. Unexpected flexibility in an evolutionarily conserved protein-RNA interaction: genetic analysis of the Sm binding site. EMBO J. 9:2555-2561. 26. Konarska, M. M., and P. A. Sharp. 1987. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell 49:763-774. 27. Korf, G. M., I. W. Botros, and W. E. Stumph. 1988. Development- and tissue-specific expression of U4 small nuclear RNA genes. Mol. Cell. Biol. 8:5566-5569. 28. Krainer, A. R. 1988. Pre-mRNA splicing by complementation with purified human Ul, U2, U4/U6, and U5 snRNPs. Nucleic Acids Res. 16:9415-9429. 29. Krol, A., H. Gallinaro, E. Lazar, M. Jacob, and C. Branlant. 1981. The nuclear 5S RNAs from chicken, rat, and man. U5 RNAs are encoded by multiple genes. Nucleic Acids Res. 9:769-787. 30. Lamm, G. M., B. J. Blencowe, B. S. Sproat, A. M. Iribarren, U. Ryder, and A. I. Lamond. 1991. Antisense probes containing 2-amino-adenosine allow efficient depletion of U5 snRNP from HeLa splicing extracts. Nucleic Acids Res. 19:3193-3198. 31. Lerner, E. A., M. R. Lerner, C. A. Janeway, and J. A. Steitz. 1981. Monoclonal antibodies to nucleic acid-containing cellular constituents: probes for molecular biology and autoimmune disease. Proc. Natl. Acad. Sci. USA 78:2737-2741. 32. Lerner, M. R., J. A. Boyle, J. A. Hardin, and J. A. Steitz. 1981. Two novel classes of small ribonucleoproteins detected by

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antibodies associated with lupus erythematosis. Science 211: 400-402. 33. Lossky, M., G. J. Anderson, S. P. Jackson, and J. Beggs. 1987. Identification of a yeast snRNP protein and detection of snRNPsnRNP interactions. Cell 51:1019-1026. 34. Lund, E., C. J. Bostock, and J. E. Dahlberg. 1987. The transcription of Xenopus laevis embryonic Ul snRNA genes changes when oocytes mature into eggs. Genes Dev. 1:47-56. 35. Lund, E., and J. E. Dahlberg. 1987. Differential accumulation of Ul and U4 small nuclear RNAs during Xenopus development. Genes Dev. 1:39-46. 36. Lund, E., B. Kahan, and J. E. Dahlberg. 1985. Differential control of Ul small nuclear RNA expression during mouse development. Science 229:1271-1274. 37. Martin, F. H., and I. Tinoco, Jr. 1980. DNA-RNA hybrid duplexes containing oligo(dA:rU) sequences are exceptionally unstable and may facilitate termination of transcription. Nucleic Acids Res. 8:2295-2299. 38. Melton, D. A., P. A. Krieg, M. R. Rebagliati, T. Maniatis, K. Zinn, and M. R. Green. 1984. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12:7035-7056. 39. Mimori, T., M. Hinterberger, I. Pettersson, and J. A. Steitz. 1984. Autoantibodies to the U2 small nuclear ribonucleoprotein in a patient with scleroderma-polymyositis overlap syndrome. J. Biol. Chem. 259:560-565. 40. Montzka, K. A., and J. A. Steitz. 1988. Additional low-abundance human small nuclear ribonucleoproteins: Ull, U12, etc. Proc. Natl. Acad. Sci. USA 85:8885-8889. 41. Newman, A., and C. Norman. 1991. Mutations in yeast U5 snRNA alter the specificity of 5' splice site cleavage. Cell 65:115-124. 42. Noble, J. C. S., Z.-Q. Pan, C. Prives, and J. L. Manley. 1987. Splicing of SV40 early pre-mRNA to large T and small t mRNAs utilizes different patterns of lariat branch sites. Cell 50:227-236. 42a.Okano, Y. Personal communication. 43. Parker, R., P. G. Siliciano, and C. Guthrie. 1987. Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNP. Cell 49:229-239. 44. Patterson, B., and C. Guthrie. 1987. An essential yeast snRNA with a U5-like domain is required for splicing in vivo. Cell

49:613-624. 45. Pinto, A. L., and J. A. Steitz. 1989. The mammalian analogue of the yeast PRP8 splicing protein is present in the U4/5/6 small nuclear ribonucleoprotein particle and the spliceosome. Proc. Natl. Acad. Sci. USA 86:8742-8746. 46. Reddy, R., and H. Busch. 1988. Small nuclear RNAs: RNA sequences, structure, and modifications, p. 1-37. In M. L. Birnstiel (ed.), Structure and function of major and minor small nuclear ribonucleoprotein particles. Springer-Verlag KG, Berlin. 47. Reidel, N., J. A. Wise, H. Swerdlow, A. Mak, and C. Guthrie. 1986. Small nuclear RNAs from Saccharomyces cerevisiae: unexpected diversity in abundance, size, and molecular complexity. Proc. Natl. Acad. Sci. USA 83:8097-8101. 48. Reuter, R., and R. Luhrmann. 1986. Immunization of mice with purified Ul small nuclear ribonucleoprotein (RNP) induces a pattern of antibody specificities characteristic of the anti-Sm and anti-RNP autoimmune response of patients with lupus erythematosus, as measured by monoclonal antibodies. Proc. Natl. Acad. Sci. USA 83:8689-8693. 49. Ruby, S. W., and J. Abelson. 1988. An early hierarchic role of Ul small nuclear ribonucleoprotein in spliceosome assembly. Science 242:1028-1035. 50. Scherly, D., W. Boelens, N. A. Dathan, W. J. VenrooU, and I. W. Mattaj. 1990. Major determinants of the specificity of interaction between small nuclear ribonucleoproteins UlA and U2B" and their cognate RNAs. Nature (London) 345:502-506. 51. Seraphin, B., N. Abovich, and M. Rosbash. 1991. Genetic depletion indicates a late role for U5 snRNP during in vitro spliceosome assembly. Nucleic Acids Res. 19:3857-3860. 52. Silberklang, M., A. Gillum, and U. RajBhandary. 1979. Use of in

746

53. 54.

55. 56. 57.

SONTHEIMER AND STEITZ vitro 32P labeling in the sequence analysis of nonradioactive tRNAs. Methods Enzymol. 59:58-109. Solnick, D. 1985. Trans splicing of mRNA precursors. Cell 42:157-164. Steitz, J. A., D. L. Black, V. Gerke, K. A. Parker, A. Kramer, D. Frendewey, and W. Keller. 1988. Functions of the abundant U snRNPs, p. 115-154. In M. L. Birnstiel (ed.), Structure and function of major and minor small nuclear ribonucleoprotein particles. Springer-Verlag KG, Berlin. Strauss, E. J., and C. Guthrie. 1991. A cold-sensitive mRNA splicing mutant is a member of the RNA helicase gene family. Genes Dev. 5:629-641. Tollervey, D. 1987. High complexity of small nuclear RNAs in fungi and plants. J. Mol. Biol. 196:355-361. Winkelmann, G., M. Bach, and R. Luhrmann. 1989. Evidence from complementation assays in vitro that U5 snRNP is re-

MOL. CELL. BIOL.

quired for both steps of mRNA splicing. EMBO J. 8:3105-3112. 58. Wise, J. A., and A. M. Weiner. 1981. The small nuclear RNAs of the cellular slime mold Dictyostelium discoideum: isolation and characterization. J. Biol. Chem. 256:956-963. 59. Wu, J., and J. L. Manley. 1989. Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. Genes Dev. 3:1553-1561. 60. Zaug, A. J., J. R. Kent, and T. R. Cech. 1984. A labile phosphodiester bond at the ligation junction in a circular intervening sequence RNA. Science 224:574-578. 61. Zhuang, Y., and A. M. Weiner. 1986. A compensatory base change in Ul snRNA suppresses a 5' splice site mutation. Cell 46:827-835. 62. Zhuang, Y., and A. M. Weiner. 1989. A compensatory base change in human U2 snRNA can suppress a branch site mutation. Genes Dev. 3:1545-1552.