Rds3p Is Required for Stable U2 snRNP Recruitment to the Splicing Apparatus

Abstract

Rds3p is a well-conserved 12-kDa protein with five CxxC zinc fingers that has been implicated in the activation of certain drug transport genes and in the pre-mRNA splicing pathway. Here we show that Rds3p resides in the yeast spliceosome and is essential for splicing in vitro. Rds3p purified from yeast stably associates with at least five U2 snRNP proteins, Cus1p, Hsh49p, Hsh155p, Rse1p, and Ist3p/Snu17p, and with the Yra1p RNA export factor. A mutation upstream of the first Rds3p zinc finger causes the conditional release of the putative branchpoint nucleotide binding protein, Ist3p/Snu17p, and weakens Rse1p interaction with the Rds3p complex. The resultant U2 snRNP particle migrates exceptionally slowly in polyacrylamide gels, suggestive of a disorganized structure. U2 snRNPs depleted of Rds3p fail to form stable prespliceosomes, although U2 snRNA stability is not affected. Metabolic depletion of Yra1p blocks cell growth but not splicing, suggesting that Yra1p association with Rds3p relates to Yra1p's role in RNA trafficking. Together these data establish Rds3p as an essential component of the U2 snRNP SF3b complex and suggest a new link between the nuclear processes of pre-mRNA splicing and RNA export.

ACKNOWLEDGMENTS

We are grateful to Bert Lynn and the UK Mass Spectroscopy Facility for proteomic analysis of the Rds3p complex. We thank the Hurt lab for generously providing the yra1::HIS3 knockout strain and plasmids containing the GAL1 and protein A fusions to YRA1. Bernard Turcotte is acknowledged for discussions while this work was in progress. We thank Martha Peterson for her helpful comments on the manuscript and Kevin Vincent and Georgia Zeigler for reagents and technical assistance, respectively.

This work was sponsored by NIH award GM42476 to B.C.R. Proteomic analysis of the Rds3p complex was supported by the Kentucky NSF EPS-0132295 award.