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

Published online by Cambridge University Press:  23 August 2002

SHELLY-ANN JAMES
Affiliation:
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, USA
WILLIAM TURNER
Affiliation:
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, USA
BEATE SCHWER
Affiliation:
Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, USA
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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.

Type
Research Article
Copyright
© 2002 RNA Society

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