How Slu7 and Prp18 cooperate in the second step of yeast pre-mRNA splicing.
Slu7 and Prp18 act in concert during the second step of yeast pre-mRNA splicing. Here we show that the 382-amino-acid Slu7 protein contains two functionally important domains: a zinc knuckle (122CRNCGEAGHKEKDC135) and a Prp18-interaction domain (215EIELMKLELY224). Alanine cluster mutations of 215EIE217 and 221LELY224 abrogated Slu7 binding to Prp18 in a two-hybrid assay and in vitro, and elicited temperature-sensitive growth phenotypes in vivo. Yet, the mutations had no impact on Slu7 function in pre-mRNA splicing in vitro. Single alanine mutations of zinc knuckle residues Cys122, His130, and Cys135 had no effect on cell growth, but caused Slu7 function during pre-mRNA splicing in vitro to become dependent on Prp18. Specifically, zinc knuckle mutants required Prp18 in order to bind to the spliceosome. Compound mutations in both Slu7 domains (e.g., C122A-EIE, H130A-EIE, and C135A-EIE) were lethal in vivo and abolished splicing in vitro, suggesting that the physical interaction between Slu7 and Prp18 is important for cooperation in splicing. Depletion/reconstitution studies coupled with immunoprecipitations suggest that second step factors are recruited to the spliceosome in the following order: Slu7 --> Prp18 --> Prp22. All three proteins are released from the spliceosome after step 2 concomitant with release of mature mRNA.
James
Shelly-Ann
SA
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, USA.
Turner
William
W
Schwer
Beate
B
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D053487:DEAD-box RNA Helicases; D005656:Fungal Proteins / Q000378:metabolism; D005800:Genes, Fungal; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins; D017434:Protein Structure, Tertiary; D020365:RNA Helicases; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D017384:Sequence Deletion; D017381:Spliceosomes / Q000378:metabolism
D005656:Fungal Proteins; D009687:Nuclear Proteins; C080779:PRP18 protein, S cerevisiae; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D016601:RNA-Binding Proteins; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C095479:SLU7 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C067135:PRP22 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases