An unanticipated early function of DEAD-box ATPase Prp28 during commitment to splicing is modulated by U5 snRNP protein Prp8.
The stepwise assembly of the highly dynamic spliceosome is guided by RNA-dependent ATPases of the DEAD-box family, whose regulation is poorly understood. In the canonical assembly model, the U4/U6.U5 triple snRNP binds only after joining of the U1 and, subsequently, U2 snRNPs to the intron-containing pre-mRNA. Catalytic activation requires the exchange of U6 for U1 snRNA at the 5' splice site, which is promoted by the DEAD-box protein Prp28. Because Prp8, an integral U5 snRNP protein, is thought to be a central regulator of DEAD-box proteins, we conducted a targeted search in Prp8 for cold-insensitive suppressors of a cold-sensitive Prp28 mutant, prp28-1. We identified a cluster of suppressor mutations in an N-terminal bromodomain-like sequence of Prp8. To identify the precise defect in prp28-1 strains that is suppressed by the Prp8 alleles, we analyzed spliceosome assembly in vivo and in vitro. Surprisingly, in the prp28-1 strain, we observed a block not only to spliceosome activation but also to one of the earliest steps of assembly, formation of the ATP-independent commitment complex 2 (CC2). The Prp8 suppressor partially corrected both the early assembly and later activation defects of prp28-1, supporting a role for this U5 snRNP protein in both the ATP-independent and ATP-dependent functions of Prp28. We conclude that the U5 snRNP has a role in the earliest events of assembly, prior to its stable incorporation into the spliceosome.
Argenta M
AM
Price
Janina
J
Görnemann
Christine
C
Guthrie
David A
DA
Brow
D000255:Adenosine Triphosphate / Q000494:pharmacology; D017398:Alternative Splicing / Q000502:physiology*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000502:physiology; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055503:Protein Multimerization / Q000187:drug effects / Q000502:physiology; D012342:RNA, Small Nuclear / Q000502:physiology; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000187:drug effects / Q000502:physiology
C077018:PRP8 protein, S cerevisiae; D012342:RNA, Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C405485:U5 small nuclear RNA; D000255:Adenosine Triphosphate; C088939:PRP28 protein, S cerevisiae; D053487:DEAD-box RNA Helicases
Prp28; Prp8; commitment complex; pre-mRNA splicing; pre-spliceosome