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Molecular and Cellular Biology, November 2009, p. 5671-5678, Vol. 29, No. 21
0270-7306/09/$08.00+0     doi:10.1128/MCB.00773-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Cwc25 Is a Novel Splicing Factor Required after Prp2 and Yju2 To Facilitate the First Catalytic Reaction{triangledown}

Ying-Fang Chiu,1,{dagger} Yen-Chi Liu,1,{dagger} Ting-Wei Chiang,1,2 Tzu-Chi Yeh,1 Chi-Kang Tseng,1,3 Nan-Ying Wu,1 and Soo-Chen Cheng1,3*

Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 115, Republic of China,1 Faculty of Life Science and Institute of Genomic Science, National Yang-Ming University, Taipei, Taiwan 112, Republic of China,2 Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan 112, Republic of China3

Received 15 June 2009/ Returned for modification 10 July 2009/ Accepted 17 August 2009

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 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.

* Corresponding author. Mailing address: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 115, Republic of China. Phone: 886-2-2789-9200. Fax: 886-2-2788-3296. E-mail: mbscc{at}ccvax.sinica.edu.tw

{triangledown} Published ahead of print on 24 August 2009.

{dagger} Equal contribution.

Molecular and Cellular Biology, November 2009, p. 5671-5678, Vol. 29, No. 21
0270-7306/09/$08.00+0     doi:10.1128/MCB.00773-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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