Spp382p interacts with multiple yeast splicing factors, including possible regulators of Prp43 DExD/H-Box protein function.
Prp43p catalyzes essential steps in pre-mRNA splicing and rRNA biogenesis. In splicing, Spp382p stimulates the Prp43p helicase to dissociate the postcatalytic spliceosome and, in some way, to maintain the integrity of the spliceosome assembly. Here we present a dosage interference assay to identify Spp382p-interacting factors by screening for genes that when overexpressed specifically inhibit the growth of a conditional lethal prp38-1 spliceosome assembly mutant in the spp382-1 suppressor background. Identified, among others, are genes encoding the established splicing factors Prp8p, Prp9p, Prp11p, Prp39p, and Yhc1p and two poorly characterized proteins with possible links to splicing, Sqs1p and Cwc23p. Sqs1p copurifies with Prp43p and is shown to bind Prp43p and Spp382p in the two-hybrid assay. Overexpression of Sqs1p blocks pre-mRNA splicing and inhibits Prp43p-dependent steps in rRNA processing. Increased Prp43p levels buffer Sqs1p cytotoxicity, providing strong evidence that the Prp43p DExD/H-box protein is a target of Sqs1p. Cwc23p is the only known yeast splicing factor with a DnaJ motif characteristic of Hsp40-like chaperones. We show that similar to SPP382, CWC23 activity is critical for efficient pre-mRNA splicing and intron metabolism yet, surprisingly, this activity does not require the canonical DnaJ/Hsp40 motif. These and related data establish the value of this dosage interference assay for finding genes that alter cellular splicing and define Sqs1p and Cwc23p as prospective modulators of Spp382p-stimuated Prp43p function.
Pandit
Shatakshi
S
Department of Biology, University of Kentucky, Lexington, Kentucky 40506-0225, USA.
Paul
Sudakshina
S
Zhang
Li
L
Chen
Min
M
Durbin
Nicole
N
Harrison
Susan M W
SM
Rymond
Brian C
BC
D017398:Alternative Splicing / Q000502:physiology; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics / Q000502:physiology; D018628:Gene Dosage; D015966:Gene Expression Regulation, Fungal; D008954:Models, Biological; D011485:Protein Binding; D012322:RNA Precursors / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D013489:Suppression, Genetic / Q000502:physiology
D012322:RNA Precursors; D016601:RNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; C507652:Spp382 protein, S cerevisiae; C496127:ERG9 protein, S cerevisiae; D013186:Farnesyl-Diphosphate Farnesyltransferase; C109034:PRP43 protein, S cerevisiae; D053487:DEAD-box RNA Helicases