Cwc25 is a novel splicing factor required after Prp2 and Yju2 to facilitate the first catalytic reaction.
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.
Chiu
Ying-Fang
YF
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 115, Republic of China.
Liu
Yen-Chi
YC
Chiang
Ting-Wei
TW
Yeh
Tzu-Chi
TC
Tseng
Chi-Kang
CK
Wu
Nan-Ying
NY
Cheng
Soo-Chen
SC
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
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
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