Distinct domains of splicing factor Prp8 mediate different aspects of spliceosome activation.
Prp8 is the largest and most highly conserved protein in the spliceosome yet its mechanism of function is poorly understood. Our previous studies implicate Prp8 in control of spliceosome activation for the first catalytic step of splicing, because substitutions in five distinct regions (a-e) of Prp8 suppress a cold-sensitive block to activation caused by a mutation in U4 RNA. Catalytic activation of the spliceosome is thought to require unwinding of the U1 RNA/5' splice site and U4/U6 RNA helices by the Prp28 and Prp44/Brr2 DExD/H-box helicases, respectively. Here we show that mutations in regions a, d, and e of Prp8 exhibit allele-specific genetic interactions with mutations in Prp28, Prp44/Brr2, and U6 RNA, respectively. These results indicate that Prp8 coordinates multiple processes in spliceosome activation and enable an initial correlation of Prp8 structure and function. Furthermore, additional genetic interactions with U4-cs1 support a two-state model for this RNA conformational switch and implicate another splicing factor, Prp31, in Prp8-mediated spliceosome activation.
Kuhn
Andreas N
AN
Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, WI 53706-1532, USA.
Reichl
Elizabeth M
EM
Brow
David A
DA
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D003080:Cold Temperature; D053487:DEAD-box RNA Helicases; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000378:metabolism*
D005656:Fungal Proteins; C077018:PRP8 protein, S cerevisiae; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D012097:Repressor Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C405482:U4 small nuclear RNA; D012316:RNA Nucleotidyltransferases; C104158:BRR2 protein, S cerevisiae; C088939:PRP28 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases