Abstract
U4 small nuclear RNA (snRNA) is essential for pre-mRNA splicing, although its role is not yet clear. On the basis of a model structure (C. Guthrie and B. Patterson, Annu. Rev. Genet. 22:387-419, 1988), the molecule can be thought of as having six domains: stem II, 5' stem-loop, stem I, central region, 3' stem-loop, and 3'-terminal region. We have carried out extensive mutagenesis of the yeast U4 snRNA gene (SNR14) and have obtained information on the effect of mutations at 105 of its 160 nucleotides. Fifteen critical residues in the U4 snRNA have been identified in four domains: stem II, the 5' stem-loop, stem I, and the 3'-terminal region. These domains have been shown previously to be insensitive to oligonucleotide-directed RNase H cleavage (Y. Xu, S. Petersen-Bjørn, and J. D. Friesen, Mol. Cell. Biol. 10:1217-1225, 1990), suggesting that they are involved in intra- or intermolecular interactions. Stem II, a region that base pairs with U6 snRNA, is the most sensitive to mutation of all U4 snRNA domains. In contrast, stem I is surprisingly insensitive to mutational change, which brings into question its role in base pairing with U6 snRNA. All mutations in the putative Sm site of U4 snRNA yield a lethal or conditional-lethal phenotype, indicating that this region is important functionally. Only two nucleotides in the 5' stem-loop are sensitive to mutation; most of this domain can tolerate point mutations or small deletions. The 3' stem-loop, while essential, is very tolerant of change. A large portion of the central domain can be removed or expanded with only minor effects on phenotype, suggesting that it has little function of its own. Analysis of conditional mutations in stem II and stem I indicates that although these single-base changes do not have a dramatic effect on U4 snRNA stability, they are defective in RNA splicing in vivo and in vitro, as well as in spliceosome assembly. These results are discussed in the context of current knowledge of the interactions involving U4 snRNA.
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Selected References
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- Abovich N., Legrain P., Rosbash M. The yeast PRP6 gene encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein, and the PRP9 gene encodes a protein required for U2 snRNP binding. Mol Cell Biol. 1990 Dec;10(12):6417–6425. doi: 10.1128/mcb.10.12.6417. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ares M., Jr, Igel A. H. Lethal and temperature-sensitive mutations and their suppressors identify an essential structural element in U2 small nuclear RNA. Genes Dev. 1990 Dec;4(12A):2132–2145. doi: 10.1101/gad.4.12a.2132. [DOI] [PubMed] [Google Scholar]
- Banroques J., Abelson J. N. PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle. Mol Cell Biol. 1989 Sep;9(9):3710–3719. doi: 10.1128/mcb.9.9.3710. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Berget S. M., Robberson B. L. U1, U2, and U4/U6 small nuclear ribonucleoproteins are required for in vitro splicing but not polyadenylation. Cell. 1986 Aug 29;46(5):691–696. doi: 10.1016/0092-8674(86)90344-2. [DOI] [PubMed] [Google Scholar]
- Bindereif A., Wolff T., Green M. R. Discrete domains of human U6 snRNA required for the assembly of U4/U6 snRNP and splicing complexes. EMBO J. 1990 Jan;9(1):251–255. doi: 10.1002/j.1460-2075.1990.tb08102.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bjørn S. P., Soltyk A., Beggs J. D., Friesen J. D. PRP4 (RNA4) from Saccharomyces cerevisiae: its gene product is associated with the U4/U6 small nuclear ribonucleoprotein particle. Mol Cell Biol. 1989 Sep;9(9):3698–3709. doi: 10.1128/mcb.9.9.3698. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Black D. L., Chabot B., Steitz J. A. U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing. Cell. 1985 Oct;42(3):737–750. doi: 10.1016/0092-8674(85)90270-3. [DOI] [PubMed] [Google Scholar]
- Black D. L., Steitz J. A. Pre-mRNA splicing in vitro requires intact U4/U6 small nuclear ribonucleoprotein. Cell. 1986 Aug 29;46(5):697–704. doi: 10.1016/0092-8674(86)90345-4. [DOI] [PubMed] [Google Scholar]
- Blencowe B. J., Sproat B. S., Ryder U., Barabino S., Lamond A. I. Antisense probing of the human U4/U6 snRNP with biotinylated 2'-OMe RNA oligonucleotides. Cell. 1989 Nov 3;59(3):531–539. doi: 10.1016/0092-8674(89)90036-6. [DOI] [PubMed] [Google Scholar]
- Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
- Bonneaud N., Ozier-Kalogeropoulos O., Li G. Y., Labouesse M., Minvielle-Sebastia L., Lacroute F. A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast. 1991 Aug-Sep;7(6):609–615. doi: 10.1002/yea.320070609. [DOI] [PubMed] [Google Scholar]
- Bordonné R., Banroques J., Abelson J., Guthrie C. Domains of yeast U4 spliceosomal RNA required for PRP4 protein binding, snRNP-snRNP interactions, and pre-mRNA splicing in vivo. Genes Dev. 1990 Jul;4(7):1185–1196. doi: 10.1101/gad.4.7.1185. [DOI] [PubMed] [Google Scholar]
- Bringmann P., Appel B., Rinke J., Reuter R., Theissen H., Lührmann R. Evidence for the existence of snRNAs U4 and U6 in a single ribonucleoprotein complex and for their association by intermolecular base pairing. EMBO J. 1984 Jun;3(6):1357–1363. doi: 10.1002/j.1460-2075.1984.tb01977.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cheng S. C., Abelson J. Spliceosome assembly in yeast. Genes Dev. 1987 Nov;1(9):1014–1027. doi: 10.1101/gad.1.9.1014. [DOI] [PubMed] [Google Scholar]
- Datta B., Weiner A. M. Genetic evidence for base pairing between U2 and U6 snRNA in mammalian mRNA splicing. Nature. 1991 Aug 29;352(6338):821–824. doi: 10.1038/352821a0. [DOI] [PubMed] [Google Scholar]
- Guthrie C. Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. Science. 1991 Jul 12;253(5016):157–163. doi: 10.1126/science.1853200. [DOI] [PubMed] [Google Scholar]
- Guthrie C., Patterson B. Spliceosomal snRNAs. Annu Rev Genet. 1988;22:387–419. doi: 10.1146/annurev.ge.22.120188.002131. [DOI] [PubMed] [Google Scholar]
- Hashimoto C., Steitz J. A. U4 and U6 RNAs coexist in a single small nuclear ribonucleoprotein particle. Nucleic Acids Res. 1984 Apr 11;12(7):3283–3293. doi: 10.1093/nar/12.7.3283. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hausner T. P., Giglio L. M., Weiner A. M. Evidence for base-pairing between mammalian U2 and U6 small nuclear ribonucleoprotein particles. Genes Dev. 1990 Dec;4(12A):2146–2156. doi: 10.1101/gad.4.12a.2146. [DOI] [PubMed] [Google Scholar]
- Hu J., Xu Y., Schappert K., Harrington T., Wang A., Braga R., Mogridge J., Friesen J. D. Mutational analysis of the PRP4 protein of Saccharomyces cerevisiae suggests domain structure and snRNP interactions. Nucleic Acids Res. 1994 May 11;22(9):1724–1734. doi: 10.1093/nar/22.9.1724. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jones M. H., Guthrie C. Unexpected flexibility in an evolutionarily conserved protein-RNA interaction: genetic analysis of the Sm binding site. EMBO J. 1990 Aug;9(8):2555–2561. doi: 10.1002/j.1460-2075.1990.tb07436.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lamond A. I., Konarska M. M., Grabowski P. J., Sharp P. A. Spliceosome assembly involves the binding and release of U4 small nuclear ribonucleoprotein. Proc Natl Acad Sci U S A. 1988 Jan;85(2):411–415. doi: 10.1073/pnas.85.2.411. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liao X. L., Kretzner L., Seraphin B., Rosbash M. Universally conserved and yeast-specific U1 snRNA sequences are important but not essential for U1 snRNP function. Genes Dev. 1990 Oct;4(10):1766–1774. doi: 10.1101/gad.4.10.1766. [DOI] [PubMed] [Google Scholar]
- Lin R. J., Newman A. J., Cheng S. C., Abelson J. Yeast mRNA splicing in vitro. J Biol Chem. 1985 Nov 25;260(27):14780–14792. [PubMed] [Google Scholar]
- Madhani H. D., Bordonné R., Guthrie C. Multiple roles for U6 snRNA in the splicing pathway. Genes Dev. 1990 Dec;4(12B):2264–2277. doi: 10.1101/gad.4.12b.2264. [DOI] [PubMed] [Google Scholar]
- Madhani H. D., Guthrie C. A novel base-pairing interaction between U2 and U6 snRNAs suggests a mechanism for the catalytic activation of the spliceosome. Cell. 1992 Nov 27;71(5):803–817. doi: 10.1016/0092-8674(92)90556-r. [DOI] [PubMed] [Google Scholar]
- Maniatis T., Reed R. The role of small nuclear ribonucleoprotein particles in pre-mRNA splicing. Nature. 1987 Feb 19;325(6106):673–678. doi: 10.1038/325673a0. [DOI] [PubMed] [Google Scholar]
- Munholland J. M., Kelly J. K., Wildeman A. G. DNA sequences required for yeast actin gene transcription do not include conserved CCAAT motifs. Nucleic Acids Res. 1990 Oct 25;18(20):6061–6068. doi: 10.1093/nar/18.20.6061. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Myslinski E., Branlant C. A phylogenetic study of U4 snRNA reveals the existence of an evolutionarily conserved secondary structure corresponding to 'free' U4 snRNA. Biochimie. 1991 Jan;73(1):17–28. doi: 10.1016/0300-9084(91)90069-d. [DOI] [PubMed] [Google Scholar]
- Newman A. J., Norman C. U5 snRNA interacts with exon sequences at 5' and 3' splice sites. Cell. 1992 Feb 21;68(4):743–754. doi: 10.1016/0092-8674(92)90149-7. [DOI] [PubMed] [Google Scholar]
- Newman A., Norman C. Mutations in yeast U5 snRNA alter the specificity of 5' splice-site cleavage. Cell. 1991 Apr 5;65(1):115–123. doi: 10.1016/0092-8674(91)90413-s. [DOI] [PubMed] [Google Scholar]
- Parker R., Siliciano P. G., Guthrie C. Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA. Cell. 1987 Apr 24;49(2):229–239. doi: 10.1016/0092-8674(87)90564-2. [DOI] [PubMed] [Google Scholar]
- Percival-Smith A., Segall J. Characterization and mutational analysis of a cluster of three genes expressed preferentially during sporulation of Saccharomyces cerevisiae. Mol Cell Biol. 1986 Jul;6(7):2443–2451. doi: 10.1128/mcb.6.7.2443. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pikielny C. W., Rymond B. C., Rosbash M. Electrophoresis of ribonucleoproteins reveals an ordered assembly pathway of yeast splicing complexes. 1986 Nov 27-Dec 3Nature. 324(6095):341–345. doi: 10.1038/324341a0. [DOI] [PubMed] [Google Scholar]
- Shannon K. W., Guthrie C. Suppressors of a U4 snRNA mutation define a novel U6 snRNP protein with RNA-binding motifs. Genes Dev. 1991 May;5(5):773–785. doi: 10.1101/gad.5.5.773. [DOI] [PubMed] [Google Scholar]
- Sharp P. A. Splicing of messenger RNA precursors. Science. 1987 Feb 13;235(4790):766–771. doi: 10.1126/science.3544217. [DOI] [PubMed] [Google Scholar]
- Shuster E. O., Guthrie C. Two conserved domains of yeast U2 snRNA are separated by 945 nonessential nucleotides. Cell. 1988 Oct 7;55(1):41–48. doi: 10.1016/0092-8674(88)90007-4. [DOI] [PubMed] [Google Scholar]
- Siliciano P. G., Brow D. A., Roiha H., Guthrie C. An essential snRNA from S. cerevisiae has properties predicted for U4, including interaction with a U6-like snRNA. Cell. 1987 Aug 14;50(4):585–592. doi: 10.1016/0092-8674(87)90031-6. [DOI] [PubMed] [Google Scholar]
- Soltyk A., Tropak M., Friesen J. D. Isolation and characterization of the RNA2+, RNA4+, and RNA11+ genes of Saccharomyces cerevisiae. J Bacteriol. 1984 Dec;160(3):1093–1100. doi: 10.1128/jb.160.3.1093-1100.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Steitz J. A. "Snurps". Sci Am. 1988 Jun;258(6):56-60, 63. doi: 10.1038/scientificamerican0688-56. [DOI] [PubMed] [Google Scholar]
- Strathern J. N., Higgins D. R. Recovery of plasmids from yeast into Escherichia coli: shuttle vectors. Methods Enzymol. 1991;194:319–329. doi: 10.1016/0076-6879(91)94024-7. [DOI] [PubMed] [Google Scholar]
- Teigelkamp S., McGarvey M., Plumpton M., Beggs J. D. The splicing factor PRP2, a putative RNA helicase, interacts directly with pre-mRNA. EMBO J. 1994 Feb 15;13(4):888–897. doi: 10.1002/j.1460-2075.1994.tb06332.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vankan P., McGuigan C., Mattaj I. W. Domains of U4 and U6 snRNAs required for snRNP assembly and splicing complementation in Xenopus oocytes. EMBO J. 1990 Oct;9(10):3397–3404. doi: 10.1002/j.1460-2075.1990.tb07541.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vankan P., McGuigan C., Mattaj I. W. Roles of U4 and U6 snRNAs in the assembly of splicing complexes. EMBO J. 1992 Jan;11(1):335–343. doi: 10.1002/j.1460-2075.1992.tb05056.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wersig C., Bindereif A. Conserved domains of human U4 snRNA required for snRNP and spliceosome assembly. Nucleic Acids Res. 1990 Nov 11;18(21):6223–6229. doi: 10.1093/nar/18.21.6223. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Whittaker E., Beggs J. D. The yeast PRP8 protein interacts directly with pre-mRNA. Nucleic Acids Res. 1991 Oct 25;19(20):5483–5489. doi: 10.1093/nar/19.20.5483. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Woolford J. L., Jr Nuclear pre-mRNA splicing in yeast. Yeast. 1989 Nov-Dec;5(6):439–457. doi: 10.1002/yea.320050604. [DOI] [PubMed] [Google Scholar]
- Wu J. A., Manley J. L. Base pairing between U2 and U6 snRNAs is necessary for splicing of a mammalian pre-mRNA. Nature. 1991 Aug 29;352(6338):818–821. doi: 10.1038/352818a0. [DOI] [PubMed] [Google Scholar]
- Wu J., Manley J. L. Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. Genes Dev. 1989 Oct;3(10):1553–1561. doi: 10.1101/gad.3.10.1553. [DOI] [PubMed] [Google Scholar]
- Xu Y., Petersen-Bjørn S., Friesen J. D. The PRP4 (RNA4) protein of Saccharomyces cerevisiae is associated with the 5' portion of the U4 small nuclear RNA. Mol Cell Biol. 1990 Mar;10(3):1217–1225. doi: 10.1128/mcb.10.3.1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yang J. H., Cedergren R., Nadal-Ginard B. Catalytic activity of an RNA domain derived from the U6-U4 RNA complex. Science. 1994 Jan 7;263(5143):77–81. doi: 10.1126/science.8272868. [DOI] [PubMed] [Google Scholar]
- Yean S. L., Lin R. J. U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction. Mol Cell Biol. 1991 Nov;11(11):5571–5577. doi: 10.1128/mcb.11.11.5571. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhuang Y., Leung H., Weiner A. M. The natural 5' splice site of simian virus 40 large T antigen can be improved by increasing the base complementarity to U1 RNA. Mol Cell Biol. 1987 Aug;7(8):3018–3020. doi: 10.1128/mcb.7.8.3018. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhuang Y., Weiner A. M. A compensatory base change in human U2 snRNA can suppress a branch site mutation. Genes Dev. 1989 Oct;3(10):1545–1552. doi: 10.1101/gad.3.10.1545. [DOI] [PubMed] [Google Scholar]