Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

RNA logoLink to RNA
. 2001 Feb;7(2):182–193. doi: 10.1017/s1355838201001807

The 100-kda U5 snRNP protein (hPrp28p) contacts the 5' splice site through its ATPase site.

N Ismaïli 1, M Sha 1, E H Gustafson 1, M M Konarska 1
PMCID: PMC1370077  PMID: 11233976

Abstract

To identify splicing factors in proximity of the 5' splice site (5'SS), we followed a crosslinking profile of site-specifically modified, photoreactive RNA substrates. Upon U4/U5/U6 snRNP addition, the 5'SS RNA crosslinks in an ATP-dependent manner to U6 snRNA, an unidentified protein p27, and the 100-kDa U5 snRNP protein, a human ortholog of an ATPase/RNA helicase yPrp28p. The 5'SS:hPrp28p crosslink maps to the highly conserved TAT motif in proximity of the ATP-binding site in hPrp28p. We propose that hPrp28p acts as a helicase to unwind the 5'SS:U1 snRNA duplex, and at the same time as a 5'SS translocase, which, upon NTP-dependent conformational change, positions the 5'SS for pairing with U6 snRNA within the spliceosome. This repositioning of the 5'SS takes place regardless of whether the 5'SS is originally duplexed with U1 snRNA.

Full Text

The Full Text of this article is available as a PDF (1,010.0 KB).

Selected References

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

  1. Bird L. E., Subramanya H. S., Wigley D. B. Helicases: a unifying structural theme? Curr Opin Struct Biol. 1998 Feb;8(1):14–18. doi: 10.1016/s0959-440x(98)80004-3. [DOI] [PubMed] [Google Scholar]
  2. Collins C. A., Guthrie C. Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome. Genes Dev. 1999 Aug 1;13(15):1970–1982. doi: 10.1101/gad.13.15.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Fu X. D. The superfamily of arginine/serine-rich splicing factors. RNA. 1995 Sep;1(7):663–680. [PMC free article] [PubMed] [Google Scholar]
  6. Gottschalk A., Neubauer G., Banroques J., Mann M., Lührmann R., Fabrizio P. Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6.U5] tri-snRNP. EMBO J. 1999 Aug 16;18(16):4535–4548. doi: 10.1093/emboj/18.16.4535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gozani O., Patton J. G., Reed R. A novel set of spliceosome-associated proteins and the essential splicing factor PSF bind stably to pre-mRNA prior to catalytic step II of the splicing reaction. EMBO J. 1994 Jul 15;13(14):3356–3367. doi: 10.1002/j.1460-2075.1994.tb06638.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gross C. H., Shuman S. The nucleoside triphosphatase and helicase activities of vaccinia virus NPH-II are essential for virus replication. J Virol. 1998 Jun;72(6):4729–4736. doi: 10.1128/jvi.72.6.4729-4736.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kim D. H., Rossi J. J. The first ATPase domain of the yeast 246-kDa protein is required for in vivo unwinding of the U4/U6 duplex. RNA. 1999 Jul;5(7):959–971. doi: 10.1017/s135583829999012x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kim J. L., Morgenstern K. A., Griffith J. P., Dwyer M. D., Thomson J. A., Murcko M. A., Lin C., Caron P. R. Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure. 1998 Jan 15;6(1):89–100. doi: 10.1016/s0969-2126(98)00010-0. [DOI] [PubMed] [Google Scholar]
  11. Kim S. H., Lin R. J. Spliceosome activation by PRP2 ATPase prior to the first transesterification reaction of pre-mRNA splicing. Mol Cell Biol. 1996 Dec;16(12):6810–6819. doi: 10.1128/mcb.16.12.6810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Konarska M. M. Analysis of splicing complexes and small nuclear ribonucleoprotein particles by native gel electrophoresis. Methods Enzymol. 1989;180:442–453. doi: 10.1016/0076-6879(89)80116-8. [DOI] [PubMed] [Google Scholar]
  13. Konarska M. M. Site-specific derivatization of RNA with photocrosslinkable groups. Methods. 1999 May;18(1):22–28. doi: 10.1006/meth.1999.0753. [DOI] [PubMed] [Google Scholar]
  14. Konforti B. B., Konarska M. M. A short 5' splice site RNA oligo can participate in both steps of splicing in mammalian extracts. RNA. 1995 Oct;1(8):815–827. [PMC free article] [PubMed] [Google Scholar]
  15. Konforti B. B., Konarska M. M. U4/U5/U6 snRNP recognizes the 5' splice site in the absence of U2 snRNP. Genes Dev. 1994 Aug 15;8(16):1962–1973. doi: 10.1101/gad.8.16.1962. [DOI] [PubMed] [Google Scholar]
  16. Konforti B. B., Koziolkiewicz M. J., Konarska M. M. Disruption of base pairing between the 5' splice site and the 5' end of U1 snRNA is required for spliceosome assembly. Cell. 1993 Dec 3;75(5):863–873. doi: 10.1016/0092-8674(93)90531-t. [DOI] [PubMed] [Google Scholar]
  17. Korolev S., Hsieh J., Gauss G. H., Lohman T. M., Waksman G. Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP. Cell. 1997 Aug 22;90(4):635–647. doi: 10.1016/s0092-8674(00)80525-5. [DOI] [PubMed] [Google Scholar]
  18. Laggerbauer B., Achsel T., Lührmann R. The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro. Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4188–4192. doi: 10.1073/pnas.95.8.4188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lauber J., Fabrizio P., Teigelkamp S., Lane W. S., Hartmann E., Luhrmann R. The HeLa 200 kDa U5 snRNP-specific protein and its homologue in Saccharomyces cerevisiae are members of the DEXH-box protein family of putative RNA helicases. EMBO J. 1996 Aug 1;15(15):4001–4015. [PMC free article] [PubMed] [Google Scholar]
  20. Lüking A., Stahl U., Schmidt U. The protein family of RNA helicases. Crit Rev Biochem Mol Biol. 1998;33(4):259–296. doi: 10.1080/10409239891204233. [DOI] [PubMed] [Google Scholar]
  21. Maroney P. A., Romfo C. M., Nilsen T. W. Functional recognition of 5' splice site by U4/U6.U5 tri-snRNP defines a novel ATP-dependent step in early spliceosome assembly. Mol Cell. 2000 Aug;6(2):317–328. doi: 10.1016/s1097-2765(00)00032-0. [DOI] [PubMed] [Google Scholar]
  22. Moore M. J., Sharp P. A. Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites. Science. 1992 May 15;256(5059):992–997. doi: 10.1126/science.1589782. [DOI] [PubMed] [Google Scholar]
  23. Murray H. L., Jarrell K. A. Flipping the switch to an active spliceosome. Cell. 1999 Mar 5;96(5):599–602. doi: 10.1016/s0092-8674(00)80568-1. [DOI] [PubMed] [Google Scholar]
  24. Neugebauer K. M., Stolk J. A., Roth M. B. A conserved epitope on a subset of SR proteins defines a larger family of Pre-mRNA splicing factors. J Cell Biol. 1995 May;129(4):899–908. doi: 10.1083/jcb.129.4.899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Ono Y., Ohno M., Shimura Y. Identification of a putative RNA helicase (HRH1), a human homolog of yeast Prp22. Mol Cell Biol. 1994 Nov;14(11):7611–7620. doi: 10.1128/mcb.14.11.7611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Patton J. G., Porro E. B., Galceran J., Tempst P., Nadal-Ginard B. Cloning and characterization of PSF, a novel pre-mRNA splicing factor. Genes Dev. 1993 Mar;7(3):393–406. doi: 10.1101/gad.7.3.393. [DOI] [PubMed] [Google Scholar]
  28. Plumpton M., McGarvey M., Beggs J. D. A dominant negative mutation in the conserved RNA helicase motif 'SAT' causes splicing factor PRP2 to stall in spliceosomes. EMBO J. 1994 Feb 15;13(4):879–887. doi: 10.1002/j.1460-2075.1994.tb06331.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Reyes J. L., Gustafson E. H., Luo H. R., Moore M. J., Konarska M. M. The C-terminal region of hPrp8 interacts with the conserved GU dinucleotide at the 5' splice site. RNA. 1999 Feb;5(2):167–179. doi: 10.1017/s1355838299981785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Reyes J. L., Kois P., Konforti B. B., Konarska M. M. The canonical GU dinucleotide at the 5' splice site is recognized by p220 of the U5 snRNP within the spliceosome. RNA. 1996 Mar;2(3):213–225. [PMC free article] [PubMed] [Google Scholar]
  32. Schmid S. R., Linder P. D-E-A-D protein family of putative RNA helicases. Mol Microbiol. 1992 Feb;6(3):283–291. doi: 10.1111/j.1365-2958.1992.tb01470.x. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Schwer B., Guthrie C. A dominant negative mutation in a spliceosomal ATPase affects ATP hydrolysis but not binding to the spliceosome. Mol Cell Biol. 1992 Aug;12(8):3540–3547. doi: 10.1128/mcb.12.8.3540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schwer B., Guthrie C. PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome. Nature. 1991 Feb 7;349(6309):494–499. doi: 10.1038/349494a0. [DOI] [PubMed] [Google Scholar]
  36. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  37. Sha M., Levy T., Kois P., Konarska M. M. Probing of the spliceosome with site-specifically derivatized 5' splice site RNA oligonucleotides. RNA. 1998 Sep;4(9):1069–1082. doi: 10.1017/s1355838298980682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Siatecka M., Reyes J. L., Konarska M. M. Functional interactions of Prp8 with both splice sites at the spliceosomal catalytic center. Genes Dev. 1999 Aug 1;13(15):1983–1993. doi: 10.1101/gad.13.15.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Strauss E. J., Guthrie C. A cold-sensitive mRNA splicing mutant is a member of the RNA helicase gene family. Genes Dev. 1991 Apr;5(4):629–641. doi: 10.1101/gad.5.4.629. [DOI] [PubMed] [Google Scholar]
  42. Strauss E. J., Guthrie C. PRP28, a 'DEAD-box' protein, is required for the first step of mRNA splicing in vitro. Nucleic Acids Res. 1994 Aug 11;22(15):3187–3193. doi: 10.1093/nar/22.15.3187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Subramanya H. S., Bird L. E., Brannigan J. A., Wigley D. B. Crystal structure of a DExx box DNA helicase. Nature. 1996 Nov 28;384(6607):379–383. doi: 10.1038/384379a0. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Teigelkamp S., Mundt C., Achsel T., Will C. L., Lührmann R. The human U5 snRNP-specific 100-kD protein is an RS domain-containing, putative RNA helicase with significant homology to the yeast splicing factor Prp28p. RNA. 1997 Nov;3(11):1313–1326. [PMC free article] [PubMed] [Google Scholar]
  46. Velankar S. S., Soultanas P., Dillingham M. S., Subramanya H. S., Wigley D. B. Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism. Cell. 1999 Apr 2;97(1):75–84. doi: 10.1016/s0092-8674(00)80716-3. [DOI] [PubMed] [Google Scholar]
  47. Vidal V. P., Verdone L., Mayes A. E., Beggs J. D. Characterization of U6 snRNA-protein interactions. RNA. 1999 Nov;5(11):1470–1481. doi: 10.1017/s1355838299991355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wagner J. D., Jankowsky E., Company M., Pyle A. M., Abelson J. N. The DEAH-box protein PRP22 is an ATPase that mediates ATP-dependent mRNA release from the spliceosome and unwinds RNA duplexes. EMBO J. 1998 May 15;17(10):2926–2937. doi: 10.1093/emboj/17.10.2926. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wang Y., Wagner J. D., Guthrie C. The DEAH-box splicing factor Prp16 unwinds RNA duplexes in vitro. Curr Biol. 1998 Apr 9;8(8):441–451. doi: 10.1016/s0960-9822(98)70178-2. [DOI] [PubMed] [Google Scholar]
  50. Wassarman D. A., Steitz J. A. Interactions of small nuclear RNA's with precursor messenger RNA during in vitro splicing. Science. 1992 Sep 25;257(5078):1918–1925. doi: 10.1126/science.1411506. [DOI] [PubMed] [Google Scholar]
  51. Yao N., Hesson T., Cable M., Hong Z., Kwong A. D., Le H. V., Weber P. C. Structure of the hepatitis C virus RNA helicase domain. Nat Struct Biol. 1997 Jun;4(6):463–467. doi: 10.1038/nsb0697-463. [DOI] [PubMed] [Google Scholar]
  52. Zhou Z., Reed R. Human homologs of yeast prp16 and prp17 reveal conservation of the mechanism for catalytic step II of pre-mRNA splicing. EMBO J. 1998 Apr 1;17(7):2095–2106. doi: 10.1093/emboj/17.7.2095. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES