Skip to main content
PMC home page
RNA logoLink to RNA
. 2001 Sep;7(9):1298–1309. doi: 10.1017/s1355838201010561

The ATP requirement for U2 snRNP addition is linked to the pre-mRNA region 5' to the branch site.

C M Newnham 1, C C Query 1
PMCID: PMC1370173  PMID: 11565751

Abstract

Association of U2 snRNP with the pre-mRNA branch region is the first ATP-dependent step in spliceosome assembly. The basis of this energy dependence is not known. Previously, we identified minimal intron-derived substrates that form complexes with U2 independent of ATP. Here, we identify the intron region linked to the ATP dependence of this step by comparing these substrates to longer RNAs that recapitulate the ATP requirement. This region needed to impose ATP dependence lies immediately 5' to the branch site. Sequences ranging from 6 to 14 nt yield a near linear inhibitory effect on efficiency of complex formation with U2 snRNP, with 18 nt yielding near maximal ATP dependence. This region is not protected prior to U2 addition, and RNase H targeting of the region within nuclear extract converts an ATP-dependent substrate into an ATP-independent one. Within this region, there is no sequence specificity linked with the ATP requirement, as neither a specific sequence is needed, nor even nucleobases. These data and the results of other modifications suggest models in which the 18-nt region is a target for interactions with U2 snRNP in an ATP-bound or -activated conformation.

Full Text

The Full Text of this article is available as a PDF (860.7 KB).

Selected References

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

  1. 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]
  2. Ast G., Pavelitz T., Weiner A. M. Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction. Nucleic Acids Res. 2001 Apr 15;29(8):1741–1749. doi: 10.1093/nar/29.8.1741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berglund J. A., Chua K., Abovich N., Reed R., Rosbash M. The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC. Cell. 1997 May 30;89(5):781–787. doi: 10.1016/s0092-8674(00)80261-5. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Chen J. Y., Stands L., Staley J. P., Jackups R. R., Jr, Latus L. J., Chang T. H. Specific alterations of U1-C protein or U1 small nuclear RNA can eliminate the requirement of Prp28p, an essential DEAD box splicing factor. Mol Cell. 2001 Jan;7(1):227–232. doi: 10.1016/s1097-2765(01)00170-8. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Dalbadie-McFarland G., Abelson J. PRP5: a helicase-like protein required for mRNA splicing in yeast. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4236–4240. doi: 10.1073/pnas.87.11.4236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Das R., Zhou Z., Reed R. Functional association of U2 snRNP with the ATP-independent spliceosomal complex E. Mol Cell. 2000 May;5(5):779–787. doi: 10.1016/s1097-2765(00)80318-4. [DOI] [PubMed] [Google Scholar]
  9. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fleckner J., Zhang M., Valcárcel J., Green M. R. U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction. Genes Dev. 1997 Jul 15;11(14):1864–1872. doi: 10.1101/gad.11.14.1864. [DOI] [PubMed] [Google Scholar]
  11. Gozani O., Feld R., Reed R. Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A. Genes Dev. 1996 Jan 15;10(2):233–243. doi: 10.1101/gad.10.2.233. [DOI] [PubMed] [Google Scholar]
  12. Gozani O., Potashkin J., Reed R. A potential role for U2AF-SAP 155 interactions in recruiting U2 snRNP to the branch site. Mol Cell Biol. 1998 Aug;18(8):4752–4760. doi: 10.1128/mcb.18.8.4752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grabowski P. J., Padgett R. A., Sharp P. A. Messenger RNA splicing in vitro: an excised intervening sequence and a potential intermediate. Cell. 1984 Jun;37(2):415–427. doi: 10.1016/0092-8674(84)90372-6. [DOI] [PubMed] [Google Scholar]
  14. Ismaïli N., Sha M., Gustafson E. H., Konarska M. M. The 100-kda U5 snRNP protein (hPrp28p) contacts the 5' splice site through its ATPase site. RNA. 2001 Feb;7(2):182–193. doi: 10.1017/s1355838201001807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kanopka A., Mühlemann O., Akusjärvi G. Inhibition by SR proteins of splicing of a regulated adenovirus pre-mRNA. Nature. 1996 Jun 6;381(6582):535–538. doi: 10.1038/381535a0. [DOI] [PubMed] [Google Scholar]
  16. Kanopka A., Mühlemann O., Petersen-Mahrt S., Estmer C., Ohrmalm C., Akusjärvi G. Regulation of adenovirus alternative RNA splicing by dephosphorylation of SR proteins. Nature. 1998 May 14;393(6681):185–187. doi: 10.1038/30277. [DOI] [PubMed] [Google Scholar]
  17. Kistler A. L., Guthrie C. Deletion of MUD2, the yeast homolog of U2AF65, can bypass the requirement for sub2, an essential spliceosomal ATPase. Genes Dev. 2001 Jan 1;15(1):42–49. doi: 10.1101/gad.851301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Konarska M. M., Sharp P. A. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell. 1987 Jun 19;49(6):763–774. doi: 10.1016/0092-8674(87)90614-3. [DOI] [PubMed] [Google Scholar]
  20. Krämer A., Grüter P., Gröning K., Kastner B. Combined biochemical and electron microscopic analyses reveal the architecture of the mammalian U2 snRNP. J Cell Biol. 1999 Jun 28;145(7):1355–1368. doi: 10.1083/jcb.145.7.1355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Krämer A. Presplicing complex formation requires two proteins and U2 snRNP. Genes Dev. 1988 Sep;2(9):1155–1167. doi: 10.1101/gad.2.9.1155. [DOI] [PubMed] [Google Scholar]
  22. Krämer A. The structure and function of proteins involved in mammalian pre-mRNA splicing. Annu Rev Biochem. 1996;65:367–409. doi: 10.1146/annurev.bi.65.070196.002055. [DOI] [PubMed] [Google Scholar]
  23. Liao X. C., Colot H. V., Wang Y., Rosbash M. Requirements for U2 snRNP addition to yeast pre-mRNA. Nucleic Acids Res. 1992 Aug 25;20(16):4237–4245. doi: 10.1093/nar/20.16.4237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Libri D., Graziani N., Saguez C., Boulay J. Multiple roles for the yeast SUB2/yUAP56 gene in splicing. Genes Dev. 2001 Jan 1;15(1):36–41. doi: 10.1101/gad.852101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Michaud S., Reed R. An ATP-independent complex commits pre-mRNA to the mammalian spliceosome assembly pathway. Genes Dev. 1991 Dec;5(12B):2534–2546. doi: 10.1101/gad.5.12b.2534. [DOI] [PubMed] [Google Scholar]
  26. Moore M. J., Query C. C. Joining of RNAs by splinted ligation. Methods Enzymol. 2000;317:109–123. doi: 10.1016/s0076-6879(00)17009-0. [DOI] [PubMed] [Google Scholar]
  27. O'Day C. L., Dalbadie-McFarland G., Abelson J. The Saccharomyces cerevisiae Prp5 protein has RNA-dependent ATPase activity with specificity for U2 small nuclear RNA. J Biol Chem. 1996 Dec 27;271(52):33261–33267. doi: 10.1074/jbc.271.52.33261. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Pauling M. H., McPheeters D. S., Ares M., Jr Functional Cus1p is found with Hsh155p in a multiprotein splicing factor associated with U2 snRNA. Mol Cell Biol. 2000 Mar;20(6):2176–2185. doi: 10.1128/mcb.20.6.2176-2185.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Perriman R., Ares M., Jr ATP can be dispensable for prespliceosome formation in yeast. Genes Dev. 2000 Jan 1;14(1):97–107. [PMC free article] [PubMed] [Google Scholar]
  31. Pruzan R., Furneaux H., Lassota P., Hong G. Y., Hurwitz J. Assemblage of the prespliceosome complex with separated fractions isolated from HeLa cells. J Biol Chem. 1990 Feb 15;265(5):2804–2813. [PubMed] [Google Scholar]
  32. Query C. C., McCaw P. S., Sharp P. A. A minimal spliceosomal complex A recognizes the branch site and polypyrimidine tract. Mol Cell Biol. 1997 May;17(5):2944–2953. doi: 10.1128/mcb.17.5.2944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Raghunathan P. L., Guthrie C. A spliceosomal recycling factor that reanneals U4 and U6 small nuclear ribonucleoprotein particles. Science. 1998 Feb 6;279(5352):857–860. doi: 10.1126/science.279.5352.857. [DOI] [PubMed] [Google Scholar]
  34. Raghunathan P. L., Guthrie C. RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2. Curr Biol. 1998 Jul 16;8(15):847–855. doi: 10.1016/s0960-9822(07)00345-4. [DOI] [PubMed] [Google Scholar]
  35. Ruby S. W., Chang T. H., Abelson J. Four yeast spliceosomal proteins (PRP5, PRP9, PRP11, and PRP21) interact to promote U2 snRNP binding to pre-mRNA. Genes Dev. 1993 Oct;7(10):1909–1925. doi: 10.1101/gad.7.10.1909. [DOI] [PubMed] [Google Scholar]
  36. Schwer B. A new twist on RNA helicases: DExH/D box proteins as RNPases. Nat Struct Biol. 2001 Feb;8(2):113–116. doi: 10.1038/84091. [DOI] [PubMed] [Google Scholar]
  37. Schwer B., Gross C. H. Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing. EMBO J. 1998 Apr 1;17(7):2086–2094. doi: 10.1093/emboj/17.7.2086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schwer B., Guthrie C. A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis. EMBO J. 1992 Dec;11(13):5033–5039. doi: 10.1002/j.1460-2075.1992.tb05610.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schwer B., Meszaros T. RNA helicase dynamics in pre-mRNA splicing. EMBO J. 2000 Dec 1;19(23):6582–6591. doi: 10.1093/emboj/19.23.6582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Staley J. P., Guthrie C. An RNA switch at the 5' splice site requires ATP and the DEAD box protein Prp28p. Mol Cell. 1999 Jan;3(1):55–64. doi: 10.1016/s1097-2765(00)80174-4. [DOI] [PubMed] [Google Scholar]
  41. Staley J. P., Guthrie C. Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell. 1998 Feb 6;92(3):315–326. doi: 10.1016/s0092-8674(00)80925-3. [DOI] [PubMed] [Google Scholar]
  42. Tazi J., Daugeron M. C., Cathala G., Brunel C., Jeanteur P. Adenosine phosphorothioates (ATP alpha S and ATP tau S) differentially affect the two steps of mammalian pre-mRNA splicing. J Biol Chem. 1992 Mar 5;267(7):4322–4326. [PubMed] [Google Scholar]
  43. Wells S. E., Ares M., Jr Interactions between highly conserved U2 small nuclear RNA structures and Prp5p, Prp9p, Prp11p, and Prp21p proteins are required to ensure integrity of the U2 small nuclear ribonucleoprotein in Saccharomyces cerevisiae. Mol Cell Biol. 1994 Sep;14(9):6337–6349. doi: 10.1128/mcb.14.9.6337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wiest D. K., O'Day C. L., Abelson J. In vitro studies of the Prp9.Prp11.Prp21 complex indicate a pathway for U2 small nuclear ribonucleoprotein activation. J Biol Chem. 1996 Dec 27;271(52):33268–33276. doi: 10.1074/jbc.271.52.33268. [DOI] [PubMed] [Google Scholar]
  45. Will C. L., Lührmann R. Molecular biology. RNP remodeling with DExH/D boxes. Science. 2001 Mar 9;291(5510):1916–1917. doi: 10.1126/science.1059682. [DOI] [PubMed] [Google Scholar]
  46. Will C. L., Schneider C., MacMillan A. M., Katopodis N. F., Neubauer G., Wilm M., Lührmann R., Query C. C. A novel U2 and U11/U12 snRNP protein that associates with the pre-mRNA branch site. EMBO J. 2001 Aug 15;20(16):4536–4546. doi: 10.1093/emboj/20.16.4536. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wincott F., DiRenzo A., Shaffer C., Grimm S., Tracz D., Workman C., Sweedler D., Gonzalez C., Scaringe S., Usman N. Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucleic Acids Res. 1995 Jul 25;23(14):2677–2684. doi: 10.1093/nar/23.14.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Yan D., Ares M., Jr Invariant U2 RNA sequences bordering the branchpoint recognition region are essential for interaction with yeast SF3a and SF3b subunits. Mol Cell Biol. 1996 Mar;16(3):818–828. doi: 10.1128/mcb.16.3.818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zhang M., Green M. R. Identification and characterization of yUAP/Sub2p, a yeast homolog of the essential human pre-mRNA splicing factor hUAP56. Genes Dev. 2001 Jan 1;15(1):30–35. doi: 10.1101/gad.851701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES