title: Cwc25 is a novel splicing factor required after Prp2 and Yju2 to facilitate the first catalytic reaction.
abstract: Cwc25 has previously been identified to associate with pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex, or NTC) involved in spliceosome activation. We show here that Cwc25 is neither tightly associated with NTC nor required for spliceosome activation but is required for the first catalytic reaction. The affinity-purified spliceosome formed in Cwc25-depleted extracts contained only pre-mRNA and could be chased into splicing intermediates upon the addition of recombinant Cwc25 in an ATP-independent manner, suggesting that Cwc25 functions in the final step of the first catalytic reaction after the action of Prp2. Yju2 and a heat-resistant factor of unknown identity, HP, have previously been shown to be required for the same step of the splicing pathway. Cwc25, although resistant to heat treatment, is not sufficient to replace the function of HP, indicating that another heat-resistant factor, which we named HP-X, is involved. The requirement of Cwc25 and HP-X for the first catalytic reaction could be partially compensated for when the affinity-purified spliceosome was incubated in the presence of low concentrations of Mn(2+). These results have implications for the possible roles of Cwc25 and HP-X in facilitating juxtaposition of the 5' splice site and the branch point during the first catalytic reaction.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 115, Republic of China.
authors.forename: Ying-Fang
authors.initials: YF
authors.lastname: Chiu
authors.forename: Yen-Chi
authors.initials: YC
authors.lastname: Liu
authors.forename: Ting-Wei
authors.initials: TW
authors.lastname: Chiang
authors.forename: Tzu-Chi
authors.initials: TC
authors.lastname: Yeh
authors.forename: Chi-Kang
authors.initials: CK
authors.lastname: Tseng
authors.forename: Nan-Ying
authors.initials: NY
authors.lastname: Wu
authors.forename: Soo-Chen
authors.initials: SC
authors.lastname: Cheng
mesh_terms: D055162:Biocatalysis / Q000187:drug effects; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D006358:Hot Temperature; D008345:Manganese / Q000494:pharmacology; D046912:Multiprotein Complexes / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing / Q000187:drug effects / Q000235:genetics*; D000072260:RNA Splicing Factors; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017381:Spliceosomes / Q000187:drug effects / Q000378:metabolism
chemical_list: C544745:Cwc25 protein, S cerevisiae; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D029701:Saccharomyces cerevisiae Proteins; C075469:YJU2 protein, S cerevisiae; D008345:Manganese; C066490:PRP2 protein, S cerevisiae; D053487:DEAD-box RNA Helicases
references.citation: Curr Opin Cell Biol. 1997 Jun;9(3):320-8
references.pmid: 9159080
references.citation: Nature. 1991 Feb 7;349(6309):494-9
references.pmid: 1825134
references.citation: Genes Dev. 2005 Dec 15;19(24):2991-3003
references.pmid: 16357217
references.citation: Mol Cell Biol. 2002 Apr;22(7):2011-24
references.pmid: 11884590
references.citation: Genes Dev. 2007 May 15;21(10):1204-16
references.pmid: 17504938
references.citation: J Biol Chem. 2005 Sep 2;280(35):31190-9
references.pmid: 15994330
references.citation: Nucleic Acids Res. 2002 Feb 15;30(4):1029-37
references.pmid: 11842115
references.citation: Cell. 1998 Feb 6;92(3):315-26
references.pmid: 9476892
references.citation: J Biol Chem. 2001 Jan 5;276(1):488-94
references.pmid: 11018040
references.citation: Nucleic Acids Res. 1997 Jun 1;25(11):2146-52
references.pmid: 9153314
references.citation: EMBO J. 1994 Feb 15;13(4):879-87
references.pmid: 8112301
references.citation: Mol Cell Biol. 1993 Mar;13(3):1883-91
references.pmid: 7680101
references.citation: Mol Cell Biol. 2005 Jan;25(1):451-60
references.pmid: 15601865
references.citation: Mol Cell Biol. 1992 Nov;12(11):5197-205
references.pmid: 1406691
references.citation: J Biol Chem. 1999 Apr 2;274(14):9455-62
references.pmid: 10092627
references.citation: Science. 2008 Jun 27;320(5884):1782-4
references.pmid: 18583613
references.citation: Genes Dev. 1992 Nov;6(11):2112-24
references.pmid: 1427075
references.citation: EMBO J. 1992 Dec;11(13):5033-9
references.pmid: 1464325
references.citation: Methods Enzymol. 1990;181:89-96
references.pmid: 2199769
references.citation: Mol Cell Biol. 1998 Apr;18(4):2196-204
references.pmid: 9528791
references.citation: RNA. 1996 Jul;2(7):707-17
references.pmid: 8756413
references.citation: Proc Natl Acad Sci U S A. 1986 Apr;83(8):2387-91
references.pmid: 3517868
references.citation: Mol Cell Biol. 1993 May;13(5):2959-70
references.pmid: 8474454
references.citation: Mol Cell Biol. 1996 Dec;16(12):6810-9
references.pmid: 8943336
references.citation: Genetics. 1987 Dec;117(4):619-31
references.pmid: 3322934
references.citation: Mol Cell Biol. 2007 Aug;27(15):5403-13
references.pmid: 17515604
references.citation: RNA. 2009 Sep;15(9):1729-39
references.pmid: 19617314
references.citation: Mol Cell Biol. 2004 Dec;24(23):10101-10
references.pmid: 15542821
references.citation: Cell. 2009 Feb 20;136(4):701-18
references.pmid: 19239890
references.citation: EMBO J. 1998 Apr 1;17(7):2086-94
references.pmid: 9524130
references.citation: RNA. 2002 Aug;8(8):1068-77
references.pmid: 12212850
references.citation: RNA. 1995 Jun;1(4):375-90
references.pmid: 7493316
references.citation: Cold Spring Harb Perspect Biol. 2011 Jul 01;3(7):
references.pmid: 21441581
references.citation: RNA. 2003 Jan;9(1):138-50
references.pmid: 12554883
references.citation: Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9687-91
references.pmid: 7568198
references.citation: Mol Cell Biol. 1990 Jan;10(1):324-32
references.pmid: 2403639
references.citation: Science. 2003 Oct 10;302(5643):279-82
references.pmid: 12970570