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RNA. 2002 Aug;8(8):1068-77.

How Slu7 and Prp18 cooperate in the second step of yeast pre-mRNA splicing.

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  • 1Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, USA.

Abstract

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.

PMID:
12212850
[PubMed - indexed for MEDLINE]
PMCID:
PMC1370317
Free PMC Article
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