Protein-free spliceosomal snRNAs catalyze a reaction that resembles the first step of splicing.
Splicing of introns from mRNA precursors is a two-step reaction performed by the spliceosome, an immense cellular machine consisting of over 200 different proteins and five small RNAs (snRNAs). We previously demonstrated that fragments of two of these RNAs, U6 and U2, can catalyze by themselves a splicing-related reaction, involving one of the two substrates of the first step of splicing, the branch site substrate. Here we show that these same RNAs can catalyze a reaction between RNA sequences that resemble the 5' splice site and the branch site, the two reactants of the first step of splicing. The reaction is dependent on the sequence of the 5' splice site consensus sequence and the catalytically essential domains of U6, and thus it resembles the authentic splicing reaction. Our results demonstrate the ability of protein-free snRNAs to recognize the sequences involved in the first splicing step and to perform splicing-related catalysis between these two pre-mRNA-like substrates.
Valadkhan
Saba
S
Center for RNA Molecular Biology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA. saba.valadkhan@case.edu
Mohammadi
Afshin
A
Wachtel
Chaim
C
Manley
James L
JL
D000818:Animals; D001483:Base Sequence; D007438:Introns; D007700:Kinetics; D009154:Mutation; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000235:genetics; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012342:RNA, Small Nuclear / Q000378:metabolism*; D012260:Ribonucleases; D017381:Spliceosomes / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015003:Yeasts / Q000235:genetics
D016337:RNA, Catalytic; D012331:RNA, Fungal; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D012260:Ribonucleases