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

TitleHow Slu7 and Prp18 cooperate in the second step of yeast pre-mRNA splicing.
Publication TypeJournal Article
Year of Publication2002
AuthorsJames S-A, Turner W, Schwer B
JournalRNA
Volume8
Issue8
Pagination1068-77
Date Published2002 Aug
ISSN1355-8382
KeywordsAmino Acid Motifs, Amino Acid Sequence, Binding Sites, DEAD-box RNA Helicases, Fungal Proteins, Genes, Fungal, Models, Biological, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Nuclear Proteins, Protein Structure, Tertiary, Ribonucleoprotein, U5 Small Nuclear, Ribonucleoproteins, Small Nuclear, RNA Helicases, RNA Precursors, RNA Splicing, RNA, Fungal, RNA-Binding Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sequence Deletion, Spliceosomes
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.

Alternate JournalRNA
PubMed ID12212850
PubMed Central IDPMC1370317
Grant ListGM50288 / GM / NIGMS NIH HHS / United States

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