title: The spliceosome catalyzes debranching in competition with reverse of the first chemical reaction.
abstract: Splicing of nuclear pre-mRNA occurs via two steps of the transesterification reaction, forming a lariat intermediate and product. The reactions are catalyzed by the spliceosome, a large ribonucleoprotein complex composed of five small nuclear RNAs and numerous protein factors. The spliceosome shares a similar catalytic core structure with that of fungal group II introns, which can self-splice using the same chemical mechanism. Like group II introns, both catalytic steps of pre-mRNA splicing can efficiently reverse on the affinity-purified spliceosome. The spliceosome also catalyzes a hydrolytic spliced-exon reopening reaction as observed in group II introns, indicating a strong link in their evolutionary relationship. We show here that, by arresting splicing after the first catalytic step, the purified spliceosome can catalyze debranching of lariat-intron-exon 2. The debranching reaction, although not observed in group II introns, has similar monovalent cation preferences as those for splicing catalysis of group II introns. The debranching reaction is in competition with the reverse Step 1 reaction influenced by the ionic environment and the structure of components binding near the catalytic center, suggesting that the catalytic center of the spliceosome can switch between different conformations to direct different chemical reactions.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan 11529, Republic of China.
authors.forename: Chi-Kang
authors.initials: CK
authors.lastname: Tseng
authors.forename: Soo-Chen
authors.initials: SC
authors.lastname: Cheng
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics; D004951:Esterification; D005091:Exons; D007438:Introns; D008274:Magnesium / Q000737:chemistry; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics; D009690:Nucleic Acid Conformation; D011189:Potassium Chloride / Q000737:chemistry; D059367:RNA Cleavage; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D017381:Spliceosomes / Q000737:chemistry* / Q000235:genetics
chemical_list: C544745:Cwc25 protein, S cerevisiae; D009687:Nuclear Proteins; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; C075469:YJU2 protein, S cerevisiae; D011189:Potassium Chloride; D012316:RNA Nucleotidyltransferases; C505387:RNA debranching enzyme Dbr1, S cerevisiae; D000251:Adenosine Triphosphatases; C075669:PRP16 protein, S cerevisiae; D020365:RNA Helicases; D008274:Magnesium
keywords: catalytic core; debranching; reverse splicing; spliceosome