Splicing Functions and Global Dependency on Fission Yeast Slu7 Reveal Diversity in Spliceosome Assembly

Abstract

The multiple short introns in Schizosaccharomyces pombe genes with degenerate cis sequences and atypically positioned polypyrimidine tracts make an interesting model to investigate canonical and alternative roles for conserved splicing factors. Here we report functions and interactions of the S. pombe slu7⁺ (spslu7⁺) gene product, known from Saccharomyces cerevisiae and human in vitro reactions to assemble into spliceosomes after the first catalytic reaction and to dictate 3′ splice site choice during the second reaction. By using a missense mutant of this essential S. pombe factor, we detected a range of global splicing derangements that were validated in assays for the splicing status of diverse candidate introns. We ascribe widespread, intron-specific SpSlu7 functions and have deduced several features, including the branch nucleotide-to-3′ splice site distance, intron length, and the impact of its A/U content at the 5′ end on the intron's dependence on SpSlu7. The data imply dynamic substrate-splicing factor relationships in multiintron transcripts. Interestingly, the unexpected early splicing arrest in spslu7-2 revealed a role before catalysis. We detected a salt-stable association with U5 snRNP and observed genetic interactions with spprp1⁺, a homolog of human U5-102k factor. These observations together point to an altered recruitment and dependence on SpSlu7, suggesting its role in facilitating transitions that promote catalysis, and highlight the diversity in spliceosome assembly.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00007-13.

ACKNOWLEDGMENTS

This work was funded by a grant to UVR from Department of Biotechnology and an infrastructure grant to the Division of Biological Sciences, Indian Institute of Science, by the Department of Biotechnology. Scholarships from IISc for S.B. and from the Council of Scientific and Industrial Research for P.K., G.M., and N.V.K. are acknowledged.

We thank Rekha Nambudry, Molecular Biophysics Unit, for assistance with Prp18 domain modeling. We acknowledge Genotypic Technology Pvt., Ltd., Bangalore, India, for microarray processing and preliminary assistance with microarray data analysis. We thank N. V. Joshi of the Centre for Ecological Sciences, IISc, for guidance and input on statistical analysis of the affected and unaffected introns. We are grateful to Amar Klar for input on tetrad dissection and to the labs of Susan Forsburg, Kathleen Gould, Jef Boeke, and Tokio Tani for key S. pombe strains. We thank Ravinder Singh for providing the chimeric minigene plasmid. Discussions and critical input from Jean Beggs and Ravinder Singh during the course of this study are gratefully acknowledged.