• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of rnaThe RNA SocietyeTOC AlertsSubscriptionsJournal HomeCSHL PressRNA
RNA. Oct 1998; 4(10): 1216–1229.
PMCID: PMC1369694

PRP16, a DEAH-box RNA helicase, is recruited to the spliceosome primarily via its nonconserved N-terminal domain.

Abstract

Dynamic rearrangement of RNA structure is crucial for intron recognition and formation of the catalytic core during pre-mRNA splicing. Three of the splicing factors that contain sequence motifs characteristic of the DExD/DExH-box family of RNA-dependent ATPases (Prp16, Prp22, and the human homologue of Brr2) recently have been shown to unwind RNA duplexes in vitro, providing biochemical evidence that they may direct structural rearrangements on the spliceosome. Notably, however, the unwinding activity of these proteins is sequence nonspecific, raising the question of how their functional specificity is determined. Because the highly conserved DExD/DExH-box domain in these proteins is typically flanked by one or more nonconserved domains, we have tested the hypothesis that the nonconserved regions of Prp16 determine the functional specificity of the protein. We found that the nonconserved N-terminal domain of Prp16 is (1) essential for viability, (2) required for the nuclear localization of Prp16, and (3) capable of binding to the spliceosome specifically at the step of Prp16 function. Moreover, this domain can interact with the rest of the protein to allow trans-complementation. Based on these results, we propose that the spliceosomal target of the unwinding activity of Prp16, and possibly other DExD/DExH-box splicing factors as well, is defined by factors that specifically interact with the nonconserved domains of the protein.

Full Text

The Full Text of this article is available as a PDF (766K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Ansari A, Schwer B. SLU7 and a novel activity, SSF1, act during the PRP16-dependent step of yeast pre-mRNA splicing. EMBO J. 1995 Aug 15;14(16):4001–4009. [PMC free article] [PubMed]
  • Arenas JE, Abelson JN. Prp43: An RNA helicase-like factor involved in spliceosome disassembly. Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):11798–11802. [PMC free article] [PubMed]
  • Ares M, Jr, Weiser B. Rearrangement of snRNA structure during assembly and function of the spliceosome. Prog Nucleic Acid Res Mol Biol. 1995;50:131–159. [PubMed]
  • Bird LE, Subramanya HS, Wigley DB. Helicases: a unifying structural theme? Curr Opin Struct Biol. 1998 Feb;8(1):14–18. [PubMed]
  • Brys A, Schwer B. Requirement for SLU7 in yeast pre-mRNA splicing is dictated by the distance between the branchpoint and the 3' splice site. RNA. 1996 Jul;2(7):707–717. [PMC free article] [PubMed]
  • Burgess S, Couto JR, Guthrie C. A putative ATP binding protein influences the fidelity of branchpoint recognition in yeast splicing. Cell. 1990 Mar 9;60(5):705–717. [PubMed]
  • Burgess SM, Guthrie C. A mechanism to enhance mRNA splicing fidelity: the RNA-dependent ATPase Prp16 governs usage of a discard pathway for aberrant lariat intermediates. Cell. 1993 Jul 2;73(7):1377–1391. [PubMed]
  • Bycroft M, Hubbard TJ, Proctor M, Freund SM, Murzin AG. The solution structure of the S1 RNA binding domain: a member of an ancient nucleic acid-binding fold. Cell. 1997 Jan 24;88(2):235–242. [PubMed]
  • Chen JH, Lin RJ. The yeast PRP2 protein, a putative RNA-dependent ATPase, shares extensive sequence homology with two other pre-mRNA splicing factors. Nucleic Acids Res. 1990 Nov 11;18(21):6447–6447. [PMC free article] [PubMed]
  • Company M, Arenas J, Abelson J. Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes. Nature. 1991 Feb 7;349(6309):487–493. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Fu XD. The superfamily of arginine/serine-rich splicing factors. RNA. 1995 Sep;1(7):663–680. [PMC free article] [PubMed]
  • Fuller-Pace FV. RNA helicases: modulators of RNA structure. Trends Cell Biol. 1994 Aug;4(8):271–274. [PubMed]
  • Gee S, Krauss SW, Miller E, Aoyagi K, Arenas J, Conboy JG. Cloning of mDEAH9, a putative RNA helicase and mammalian homologue of Saccharomyces cerevisiae splicing factor Prp43. Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):11803–11807. [PMC free article] [PubMed]
  • Gross CH, Shuman S. Mutational analysis of vaccinia virus nucleoside triphosphate phosphohydrolase II, a DExH box RNA helicase. J Virol. 1995 Aug;69(8):4727–4736. [PMC free article] [PubMed]
  • Guthrie C. Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. Science. 1991 Jul 12;253(5016):157–163. [PubMed]
  • Horowitz DS, Abelson J. Stages in the second reaction of pre-mRNA splicing: the final step is ATP independent. Genes Dev. 1993 Feb;7(2):320–329. [PubMed]
  • Horowitz DS, Abelson J. A U5 small nuclear ribonucleoprotein particle protein involved only in the second step of pre-mRNA splicing in Saccharomyces cerevisiae. Mol Cell Biol. 1993 May;13(5):2959–2970. [PMC free article] [PubMed]
  • Jones MH, Frank DN, Guthrie C. Characterization and functional ordering of Slu7p and Prp17p during the second step of pre-mRNA splicing in yeast. Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9687–9691. [PMC free article] [PubMed]
  • Kim JL, Morgenstern KA, Griffith JP, Dwyer MD, Thomson JA, Murcko MA, Lin C, Caron PR. 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. [PubMed]
  • Kim SH, Smith J, Claude A, Lin RJ. The purified yeast pre-mRNA splicing factor PRP2 is an RNA-dependent NTPase. EMBO J. 1992 Jun;11(6):2319–2326. [PMC free article] [PubMed]
  • Korolev S, Hsieh J, Gauss GH, Lohman TM, 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. [PubMed]
  • Korolev S, Yao N, Lohman TM, Weber PC, Waksman G. Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SF1 and SF2 super-families of helicases. Protein Sci. 1998 Mar;7(3):605–610. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Lauber J, Fabrizio P, Teigelkamp S, Lane WS, 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]
  • Lin J, Rossi JJ. Identification and characterization of yeast mutants that overcome an experimentally introduced block to splicing at the 3' splice site. RNA. 1996 Aug;2(8):835–848. [PMC free article] [PubMed]
  • Lin RJ, Newman AJ, Cheng SC, Abelson J. Yeast mRNA splicing in vitro. J Biol Chem. 1985 Nov 25;260(27):14780–14792. [PubMed]
  • Madhani HD, Guthrie C. Dynamic RNA-RNA interactions in the spliceosome. Annu Rev Genet. 1994;28:1–26. [PubMed]
  • Madhani HD, Guthrie C. Genetic interactions between the yeast RNA helicase homolog Prp16 and spliceosomal snRNAs identify candidate ligands for the Prp16 RNA-dependent ATPase. Genetics. 1994 Jul;137(3):677–687. [PMC free article] [PubMed]
  • Manley JL, Tacke R. SR proteins and splicing control. Genes Dev. 1996 Jul 1;10(13):1569–1579. [PubMed]
  • Noble SM, Guthrie C. Identification of novel genes required for yeast pre-mRNA splicing by means of cold-sensitive mutations. Genetics. 1996 May;143(1):67–80. [PMC free article] [PubMed]
  • O'Day CL, 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. [PubMed]
  • Ohno M, Shimura Y. A human RNA helicase-like protein, HRH1, facilitates nuclear export of spliced mRNA by releasing the RNA from the spliceosome. Genes Dev. 1996 Apr 15;10(8):997–1007. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Pause A, Méthot N, Sonenberg N. The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis. Mol Cell Biol. 1993 Nov;13(11):6789–6798. [PMC free article] [PubMed]
  • Pause A, Sonenberg N. Mutational analysis of a DEAD box RNA helicase: the mammalian translation initiation factor eIF-4A. EMBO J. 1992 Jul;11(7):2643–2654. [PMC free article] [PubMed]
  • Raghunathan PL, 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. [PubMed]
  • Roy J, Kim K, Maddock JR, Anthony JG, Woolford JL., Jr The final stages of spliceosome maturation require Spp2p that can interact with the DEAH box protein Prp2p and promote step 1 of splicing. RNA. 1995 Jun;1(4):375–390. [PMC free article] [PubMed]
  • Rozen F, Edery I, Meerovitch K, Dever TE, Merrick WC, Sonenberg N. Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F. Mol Cell Biol. 1990 Mar;10(3):1134–1144. [PMC free article] [PubMed]
  • Ruby SW, Abelson J. Pre-mRNA splicing in yeast. Trends Genet. 1991 Mar;7(3):79–85. [PubMed]
  • Schena M, Picard D, Yamamoto KR. Vectors for constitutive and inducible gene expression in yeast. Methods Enzymol. 1991;194:389–398. [PubMed]
  • Schmid SR, Linder P. D-E-A-D protein family of putative RNA helicases. Mol Microbiol. 1992 Feb;6(3):283–291. [PubMed]
  • Schwer B, Gross CH. Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing. EMBO J. 1998 Apr 1;17(7):2086–2094. [PMC free article] [PubMed]
  • Schwer B, Guthrie C. PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome. Nature. 1991 Feb 7;349(6309):494–499. [PubMed]
  • 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. [PMC free article] [PubMed]
  • 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. [PMC free article] [PubMed]
  • Sikorski RS, Boeke JD. In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Methods Enzymol. 1991;194:302–318. [PubMed]
  • Staley JP, Guthrie C. Mechanical devices of the spliceosome: motors, clocks, springs, and things. Cell. 1998 Feb 6;92(3):315–326. [PubMed]
  • Strauss EJ, 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. [PubMed]
  • Subramanya HS, Bird LE, Brannigan JA, Wigley DB. Crystal structure of a DExx box DNA helicase. Nature. 1996 Nov 28;384(6607):379–383. [PubMed]
  • Teigelkamp S, McGarvey M, Plumpton M, Beggs JD. The splicing factor PRP2, a putative RNA helicase, interacts directly with pre-mRNA. EMBO J. 1994 Feb 15;13(4):888–897. [PMC free article] [PubMed]
  • Teigelkamp S, Mundt C, Achsel T, Will CL, 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]
  • Umen JG, Guthrie C. Prp16p, Slu7p, and Prp8p interact with the 3' splice site in two distinct stages during the second catalytic step of pre-mRNA splicing. RNA. 1995 Aug;1(6):584–597. [PMC free article] [PubMed]
  • Umen JG, Guthrie C. The second catalytic step of pre-mRNA splicing. RNA. 1995 Nov;1(9):869–885. [PMC free article] [PubMed]
  • Vijayraghavan U, Company M, Abelson J. Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae. Genes Dev. 1989 Aug;3(8):1206–1216. [PubMed]
  • Wang Y, Wagner JD, Guthrie C. The DEAH-box splicing factor Prp16 unwinds RNA duplexes in vitro. Curr Biol. 1998 Apr 9;8(8):441–451. [PubMed]
  • Will CL, Lührmann R. Protein functions in pre-mRNA splicing. Curr Opin Cell Biol. 1997 Jun;9(3):320–328. [PubMed]
  • Xiao SH, Manley JL. Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing. Genes Dev. 1997 Feb 1;11(3):334–344. [PubMed]
  • Xu D, Nouraini S, Field D, Tang SJ, Friesen JD. An RNA-dependent ATPase associated with U2/U6 snRNAs in pre-mRNA splicing. Nature. 1996 Jun 20;381(6584):709–713. [PubMed]
  • Yao N, Hesson T, Cable M, Hong Z, Kwong AD, Le HV, Weber PC. Structure of the hepatitis C virus RNA helicase domain. Nat Struct Biol. 1997 Jun;4(6):463–467. [PubMed]
  • 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. [PMC free article] [PubMed]

Articles from RNA are provided here courtesy of The RNA Society

Formats: