Open AccessCwc25 Is a Novel Splicing Factor Required after Prp2 and Yju2 To Facilitate the First Catalytic Reaction
Author(s) -
Ying-Fang Chiu,
Yen-Chi Liu,
Ting-Wei Chiang,
Tzu-Chi Yeh,
C. K. Tseng,
Nan-Ying Wu,
SooChen Cheng
Publication year - 2009
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.00773-09
Subject(s) - spliceosome , rna splicing , biology , splicing factor , function (biology) , recombinant dna , microbiology and biotechnology , genetics , computational biology , rna , gene
Cwc25 has previously been identified to associate with pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (n inet eenc omplex, 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 Mn2+ . 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.