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Molecular and Cellular Biology, March 1999, p. 2008-2020, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Zoi Lygerou,dagger George Christophides,Dagger and Bertrand Séraphin*

EMBL, 69117 Heidelberg, Germany

Received 12 June 1998/Returned for modification 6 November 1998/Accepted 23 November 1998

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 sad1-1 mutant, 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 beta -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.

* Corresponding author. Mailing address: EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany. Phone: 49 6221 387 486. Fax: 49 6221 387 518. E-mail: seraphin{at}embl-heidelberg.de.

dagger Present address: Imperial Cancer Research Fund, London, United Kingdom. WC2A 3PX.

Dagger Present address: Division of Genetics and Biotechnology, Department of Biology, University of Athens, Athens, Greece.

Molecular and Cellular Biology, March 1999, p. 2008-2020, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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