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.

Second-Shell Basic Residues Expand the Two-Metal-Ion Architecture of DNA and RNA Processing Enzymes

“…Instead, in the spliceosome, we noticed that a putative K1 cavity essentially identical to the one of group II intron exists between U6-snRNA residues G52, A59, G60, and U80 ( Figure 5 ; Table S2 ). Although not modeled in the current structures, a potassium ion could optimally fit this putative K1 site at least at certain steps of the catalytic cycle, which would explain the spliceosomal dependence on potassium for splicing and debranching ( Hardy et al., 1984 , Tseng and Cheng, 2013 ). In addition, Lys611 (PROCN domain of the Prp8 subunit [ Staub et al., 2004 ], Data S1 ), which stabilizes the curvature of the intron backbone (U2 and U4) proximal to the splicing junction in the C complex, and/or conserved Arg614 (Prp8), which makes contacts with the exon in the C ∗ complex ( Figures 2 and 5 ; Table S2 ), seem to constitute optimal spliceosomal counterparts of K2.…”

Intron Invasions Trace Algal Speciation and Reveal Nearly Identical Arctic and AntarcticMicromonasPopulations

“…Because IEs are always found on the coding strand, new copies have been hypothesized to arise by intron RNA transposition through reverse splicing ( Tseng and Cheng 2008 , 2013 ) into an mRNA followed by reverse transcription of the RNA to cDNA ( Verhelst et al 2013 ) and homologous recombination. At the same time, a well-accepted model for intron removal invokes reverse transcription of spliced mRNA followed by homologous recombination ( Fink 1987 ).…”

Splicing heterogeneity: separating signal from noise

“…After assembly initiation, the spliceosome can switch between different catalytic conformations that favor forward or reverse progress [ 41 ]. The splicing catalytic process is iso-energetic and driven by numerous ATPases, resulting in two transesterification processes that are both reversible in the proper ionic environment [ 63 , 64 ]. Recent single-molecule research on spliceosome assembly has revealed that almost all of the steps in splicing are reversible [ 65 , 66 ].…”