Functional interactions between Prp8, Prp18, Slu7, and U5 snRNA during the second step of pre-mRNA splicing.
After the second transesterification step of pre-mRNA splicing, the Prp22 helicase catalyzes release of spliced mRNA by disrupting contacts in the spliceosome that likely involve Prp8. Mutations at Arg1753 in Prp8, which suppress helicase-defective prp22 mutants, elicit temperature-sensitive growth phenotypes, indicating that interactions in the spliceosome involving Prp8-R1753 might be broken prematurely at 37 degrees C. Here we report that mutations in loop I of the U5 snRNA or in Prp18 can suppress the temperature-sensitive prp8-R1753 mutants. The same gain-of-function PRP18 alleles can also alleviate the growth phenotypes of multiple slu7-ts mutants, indicating a functional link between Prp8 and the second step splicing factors Prp18 and Slu7. These findings, together with the demonstration that changes at Arg1753 in Prp8 impair step 2 of pre-mRNA splicing in vitro, are consistent with a model in which (1) Arg1753 plays a role in stabilizing U5/exon interactions prior to exon joining and (2) these contacts persist until they are broken by the helicase Prp22.
Aronova
Anna
A
Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10021, USA.
Bacíková
Dagmar
D
Crotti
Luciana B
LB
Horowitz
David S
DS
Schwer
Beate
B
D002478:Cells, Cultured; D004951:Esterification; D005091:Exons / Q000235:genetics; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing / Q000235:genetics*; D000072260:RNA Splicing Factors; D012331:RNA, Fungal / Q000737:chemistry / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
D009687:Nuclear Proteins; C080779:PRP18 protein, S cerevisiae; C077018:PRP8 protein, S cerevisiae; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C095479:SLU7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C405485:U5 small nuclear RNA