A Novel Genetic Screen for snRNP Assembly Factors in Yeast Identifies a Conserved Protein, Sad1p, Also Required for Pre-mRNA Splicing

Abstract

The assembly pathway of spliceosomal snRNPs in yeast is poorly understood. We devised a screen to identify mutations blocking the assembly of newly synthesized U4 snRNA into a functional snRNP. Fifteen mutant strains failing either to accumulate the newly synthesized U4 snRNA or to assemble a U4/U6 particle were identified and categorized into 13 complementation groups. Thirteen previously identified splicing-defective prp mutants were also assayed for U4 snRNP assembly defects. Mutations in the U4/U6 snRNP components Prp3p, Prp4p, and Prp24p led to disassembly of the U4/U6 snRNP particle and degradation of the U6 snRNA, while prp17-1 and prp19-1 strains accumulated free U4 and U6 snRNA. A detailed analysis of a newly identified mutant, the sad1-1mutant, is presented. In addition to having the snRNP assembly defect, the sad1-1 mutant is severely impaired in splicing at the restrictive temperature: the RP29 pre-mRNA strongly accumulates and splicing-dependent production of β-galactosidase from reporter constructs is abolished, while extracts prepared from sad1-1 strains fail to splice pre-mRNA substrates in vitro. The sad1-1 mutant is the only splicing-defective mutant analyzed whose mutation preferentially affects assembly of newly synthesized U4 snRNA into the U4/U6 particle. SAD1 encodes a novel protein of 52 kDa which is essential for cell viability. Sad1p localizes to the nucleus and is not stably associated with any of the U snRNAs. Sad1p contains a putative zinc finger and is phylogenetically highly conserved, with homologues identified in human,Caenorhabditis elegans, Arabidospis, and Drosophila.

ACKNOWLEDGMENTS

We thank B. M. G. Luukkonen for analyzing the splicing defect of the sad1-1 mutant by primer extension, J. Venema for suggestions on immunofluorescence, and P. Lopez, M. Luukkonen, I. Mattaj, O. Puig, B. Rutz, and J. Salgado-Garrido for discussions and comments on the manuscript. We are grateful to J. Abelson and P. Legrain for prp mutant strains and to the EMBL services for their support.

This work was supported by EMBL.