title: A central role of Cwc25 in spliceosome dynamics during the catalytic phase of pre-mRNA splicing.
abstract: Splicing of precursor mRNA occurs via two consecutive steps of transesterification reaction; both require ATP and several proteins. Despite the energy requirement in the catalytic phase, incubation of the purified spliceosome under proper ionic conditions can elicit competitive reversible transesterification, debranching, and spliced-exon-reopening reactions without the necessity for ATP or other factors, suggesting that small changes in the conformational state of the spliceosome can lead to disparate chemical consequences for the substrate. We show here that Cwc25 plays a central role in modulating the conformational state of the catalytic spliceosome during normal splicing reactions. Cwc25 binds tightly to the spliceosome after the reaction and is then removed from the spliceosome, which normally requires DExD/H-box protein Prp16 and ATP hydrolysis, to allow the occurrence of the second reaction. When deprived of Cwc25, the purified first-step spliceosome catalyzes both forward and reverse splicing reactions under normal splicing conditions without requiring energy. Both reactions are inhibited when Cwc25 is added back, presumably due to the stabilization of first-step conformation. Prp16 is dispensable for the second reaction when splicing is carried out under conditions that destabilize Cwc25. We also show that the purified precatalytic spliceosome can catalyze two steps of the reaction at a low efficiency without requiring Cwc25, Slu7, or Prp18 when incubated under proper conditions. Our study reveals conformational modulation of the spliceosome by Cwc25 and Prp16 in stabilization and destabilization of first-step conformation, respectively, to facilitate the splicing process.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 115, Republic of China.
authors.forename: Chi-Kang
authors.initials: CK
authors.lastname: Tseng
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 115, Republic of China.
authors.forename: Che-Sheng
authors.initials: CS
authors.lastname: Chung
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 115, Republic of China.
authors.forename: Hsin-Chou
authors.initials: HC
authors.lastname: Chen
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 115, Republic of China.
authors.forename: Soo-Chen
authors.initials: SC
authors.lastname: Cheng
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D055162:Biocatalysis; D015966:Gene Expression Regulation, Fungal; D006868:Hydrolysis; D008954:Models, Biological; D011487:Protein Conformation; D020365:RNA Helicases / Q000235:genetics* / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing; D000072260:RNA Splicing Factors / Q000235:genetics* / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics* / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000235:genetics / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism; D017381:Spliceosomes; D013816:Thermodynamics
chemical_list: C544745:Cwc25 protein, S cerevisiae; C080779:PRP18 protein, S cerevisiae; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C095479:SLU7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C075669:PRP16 protein, S cerevisiae; D020365:RNA Helicases
keywords: Cwc25; spliceosome; splicing catalysis