pmc.ncbi.nlm.nih.gov

Modulation of 5' splice site choice in pre-messenger RNA by two distinct steps

Abstract

Ser/Arg-rich proteins (SR proteins) are essential splicing factors that commit pre-messenger RNAs to splicing and also modulate 5' splice site choice in the presence or absence of functional U1 small nuclear ribonucleoproteins (snRNPs). Here, we perturbed the U1 snRNP in HeLa cell nuclear extract by detaching the U1-specific A protein using a 2'-O-methyl oligonucleotide (L2) complementary to its binding site in U1 RNA. In this extract, the standard adenovirus substrate is spliced normally, but excess amounts of SR proteins do not exclusively switch splicing from the normal 5' splice site to a proximal site (site 125 within the adenovirus intron), suggesting that modulation of 5' splice site choice exerted by SR proteins requires integrity of the U1 snRNP. The observation that splicing does not necessarily follow U1 binding indicates that interactions between the U1 snRNP and components assembled on the 3' splice site via SR proteins may also be critical for 5' splice site selection. Accordingly, we found that SR proteins promote the binding of the U2 snRNP to the branch site and stabilize the complex formed on a 3'-half substrate in the presence or absence of functional U1 snRNPs. A novel U2/U6/3'-half substrate crosslink was also detected and promoted by SR proteins. Our results suggest that SR proteins in collaboration with the U1 snRNP function in two distinct steps to modulate 5' splice site selection.

2504

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barabino S. M., Blencowe B. J., Ryder U., Sproat B. S., Lamond A. I. Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly. Cell. 1990 Oct 19;63(2):293–302. doi: 10.1016/0092-8674(90)90162-8. [DOI] [PubMed] [Google Scholar]
  2. Crispino J. D., Blencowe B. J., Sharp P. A. Complementation by SR proteins of pre-mRNA splicing reactions depleted of U1 snRNP. Science. 1994 Sep 23;265(5180):1866–1869. doi: 10.1126/science.8091213. [DOI] [PubMed] [Google Scholar]
  3. Eperon I. C., Ireland D. C., Smith R. A., Mayeda A., Krainer A. R. Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF. EMBO J. 1993 Sep;12(9):3607–3617. doi: 10.1002/j.1460-2075.1993.tb06034.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fu X. D., Maniatis T. The 35-kDa mammalian splicing factor SC35 mediates specific interactions between U1 and U2 small nuclear ribonucleoprotein particles at the 3' splice site. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1725–1729. doi: 10.1073/pnas.89.5.1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fu X. D. Specific commitment of different pre-mRNAs to splicing by single SR proteins. Nature. 1993 Sep 2;365(6441):82–85. doi: 10.1038/365082a0. [DOI] [PubMed] [Google Scholar]
  6. Habets W. J., Hoet M. H., De Jong B. A., Van der Kemp A., Van Venrooij W. J. Mapping of B cell epitopes on small nuclear ribonucleoproteins that react with human autoantibodies as well as with experimentally-induced mouse monoclonal antibodies. J Immunol. 1989 Oct 15;143(8):2560–2566. [PubMed] [Google Scholar]
  7. Hoffman B. E., Grabowski P. J. U1 snRNP targets an essential splicing factor, U2AF65, to the 3' splice site by a network of interactions spanning the exon. Genes Dev. 1992 Dec;6(12B):2554–2568. doi: 10.1101/gad.6.12b.2554. [DOI] [PubMed] [Google Scholar]
  8. Horowitz D. S., Krainer A. R. Mechanisms for selecting 5' splice sites in mammalian pre-mRNA splicing. Trends Genet. 1994 Mar;10(3):100–106. doi: 10.1016/0168-9525(94)90233-x. [DOI] [PubMed] [Google Scholar]
  9. Kastner B., Kornstädt U., Bach M., Lührmann R. Structure of the small nuclear RNP particle U1: identification of the two structural protuberances with RNP-antigens A and 70K. J Cell Biol. 1992 Feb;116(4):839–849. doi: 10.1083/jcb.116.4.839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kohtz J. D., Jamison S. F., Will C. L., Zuo P., Lührmann R., Garcia-Blanco M. A., Manley J. L. Protein-protein interactions and 5'-splice-site recognition in mammalian mRNA precursors. Nature. 1994 Mar 10;368(6467):119–124. doi: 10.1038/368119a0. [DOI] [PubMed] [Google Scholar]
  11. Konarska M. M., Sharp P. A. Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs. Cell. 1986 Sep 12;46(6):845–855. doi: 10.1016/0092-8674(86)90066-8. [DOI] [PubMed] [Google Scholar]
  12. Krainer A. R., Conway G. C., Kozak D. Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells. Genes Dev. 1990 Jul;4(7):1158–1171. doi: 10.1101/gad.4.7.1158. [DOI] [PubMed] [Google Scholar]
  13. Lamond A. I., Sproat B., Ryder U., Hamm J. Probing the structure and function of U2 snRNP with antisense oligonucleotides made of 2'-OMe RNA. Cell. 1989 Jul 28;58(2):383–390. doi: 10.1016/0092-8674(89)90852-0. [DOI] [PubMed] [Google Scholar]
  14. Lavigueur A., La Branche H., Kornblihtt A. R., Chabot B. A splicing enhancer in the human fibronectin alternate ED1 exon interacts with SR proteins and stimulates U2 snRNP binding. Genes Dev. 1993 Dec;7(12A):2405–2417. doi: 10.1101/gad.7.12a.2405. [DOI] [PubMed] [Google Scholar]
  15. Lutz-Freyermuth C., Keene J. D., Lutz-Reyermuth C. The U1 RNA-binding site of the U1 small nuclear ribonucleoprotein (snRNP)-associated A protein suggests a similarity with U2 snRNPs. Mol Cell Biol. 1989 Jul;9(7):2975–2982. doi: 10.1128/mcb.9.7.2975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Portman D. S., Dreyfuss G. RNA annealing activities in HeLa nuclei. EMBO J. 1994 Jan 1;13(1):213–221. doi: 10.1002/j.1460-2075.1994.tb06251.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Robberson B. L., Cote G. J., Berget S. M. Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol Cell Biol. 1990 Jan;10(1):84–94. doi: 10.1128/mcb.10.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ruby S. W., Abelson J. An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly. Science. 1988 Nov 18;242(4881):1028–1035. doi: 10.1126/science.2973660. [DOI] [PubMed] [Google Scholar]
  19. Scherly D., Boelens W., van Venrooij W. J., Dathan N. A., Hamm J., Mattaj I. W. Identification of the RNA binding segment of human U1 A protein and definition of its binding site on U1 snRNA. EMBO J. 1989 Dec 20;8(13):4163–4170. doi: 10.1002/j.1460-2075.1989.tb08601.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Seiwert S. D., Steitz J. A. Uncoupling two functions of the U1 small nuclear ribonucleoprotein particle during in vitro splicing. Mol Cell Biol. 1993 Jun;13(6):3135–3145. doi: 10.1128/mcb.13.6.3135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Solnick D. Trans splicing of mRNA precursors. Cell. 1985 Aug;42(1):157–164. doi: 10.1016/s0092-8674(85)80111-2. [DOI] [PubMed] [Google Scholar]
  22. Staknis D., Reed R. SR proteins promote the first specific recognition of Pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein particle in a general splicing enhancer complex. Mol Cell Biol. 1994 Nov;14(11):7670–7682. doi: 10.1128/mcb.14.11.7670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sun Q., Mayeda A., Hampson R. K., Krainer A. R., Rottman F. M. General splicing factor SF2/ASF promotes alternative splicing by binding to an exonic splicing enhancer. Genes Dev. 1993 Dec;7(12B):2598–2608. doi: 10.1101/gad.7.12b.2598. [DOI] [PubMed] [Google Scholar]
  24. Séraphin B., Kretzner L., Rosbash M. A U1 snRNA:pre-mRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5' cleavage site. EMBO J. 1988 Aug;7(8):2533–2538. doi: 10.1002/j.1460-2075.1988.tb03101.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tarn W. Y., Steitz J. A. SR proteins can compensate for the loss of U1 snRNP functions in vitro. Genes Dev. 1994 Nov 15;8(22):2704–2717. doi: 10.1101/gad.8.22.2704. [DOI] [PubMed] [Google Scholar]
  26. Tian M., Maniatis T. A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA. Cell. 1993 Jul 16;74(1):105–114. doi: 10.1016/0092-8674(93)90298-5. [DOI] [PubMed] [Google Scholar]
  27. Wassarman D. A., Steitz J. A. A base-pairing interaction between U2 and U6 small nuclear RNAs occurs in > 150S complexes in HeLa cell extracts: implications for the spliceosome assembly pathway. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):7139–7143. doi: 10.1073/pnas.90.15.7139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Watakabe A., Tanaka K., Shimura Y. The role of exon sequences in splice site selection. Genes Dev. 1993 Mar;7(3):407–418. doi: 10.1101/gad.7.3.407. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Wu J. Y., Maniatis T. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell. 1993 Dec 17;75(6):1061–1070. doi: 10.1016/0092-8674(93)90316-i. [DOI] [PubMed] [Google Scholar]
  31. Zahler A. M., Roth M. B. Distinct functions of SR proteins in recruitment of U1 small nuclear ribonucleoprotein to alternative 5' splice sites. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2642–2646. doi: 10.1073/pnas.92.7.2642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zamore P. D., Green M. R. Biochemical characterization of U2 snRNP auxiliary factor: an essential pre-mRNA splicing factor with a novel intranuclear distribution. EMBO J. 1991 Jan;10(1):207–214. doi: 10.1002/j.1460-2075.1991.tb07937.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zuo P., Manley J. L. The human splicing factor ASF/SF2 can specifically recognize pre-mRNA 5' splice sites. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3363–3367. doi: 10.1073/pnas.91.8.3363. [DOI] [PMC free article] [PubMed] [Google Scholar]