Evidence that U2/U6 helix I promotes both catalytic steps of pre-mRNA splicing and rearranges in between these steps.
During pre-mRNA splicing, the spliceosome must configure the substrate, catalyze 5' splice site cleavage, reposition the substrate, and catalyze exon ligation. The highly conserved U2/U6 helix I, which adjoins sequences that define the reactive sites, has been proposed to configure the substrate for 5' splice site cleavage and promote catalysis. However, a role for this helix at either catalytic step has not been tested rigorously and previous observations question its role at the catalytic steps. Through a comprehensive molecular genetic study of U2/U6 helix I, we found that weakening U2/U6 helix I, but not mutually exclusive structures, compromised splicing of a substrate limited at the catalytic step of 5' splice site cleavage, providing the first compelling evidence that this helix indeed configures the substrate during 5' splice site cleavage. Further, mutations that we proved weaken only U2/U6 helix I suppressed a mutation in PRP16, a DEAH-box ATPase required after 5' splice site cleavage, providing persuasive evidence that helix I is destabilized by Prp16p and suggesting that this structure is unwound between the catalytic steps. Lastly, weakening U2/U6 helix I also compromised splicing of a substrate limited at the catalytic step of exon ligation, providing evidence that U2/U6 helix I reforms and functions during exon ligation. Thus, our data provide evidence for a fundamental and apparently dynamic role for U2/U6 helix I during the catalytic stages of splicing.
Mefford
Melissa A
MA
Committee on Genetics, Genomics and Systems Biology, The University of Chicago, Chicago, Illinois 60637, USA.
Staley
Jonathan P
JP
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D002467:Cell Nucleus / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010957:Plasmids; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012331:RNA, Fungal / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C405484:U2 small nuclear RNA; C405486:U6 small nuclear RNA; D000251:Adenosine Triphosphatases; C075669:PRP16 protein, S cerevisiae; D020365:RNA Helicases