Removal of a Single α-Tubulin Gene Intron Suppresses Cell Cycle Arrest Phenotypes of Splicing Factor Mutations in Saccharomyces cerevisiae

Abstract

Genetic and biochemical studies of Schizosaccharomyces pombe and Saccharomyces cerevisiae have identified gene products that play essential functions in both pre-mRNA splicing and cell cycle control. Among these are the conserved, Myb-related CDC5 (also known as Cef1p in S. cerevisiae) proteins. The mechanism by which loss of CDC5/Cef1p function causes both splicing and cell cycle defects has been unclear. Here we provide evidence that cell cycle arrest in a new temperature-sensitive CEF1 mutant, cef1-13, is an indirect consequence of defects in pre-mRNA splicing. Although cef1-13 cells harbor global defects in pre-mRNA splicing discovered through intron microarray analysis, inefficient splicing of the α-tubulin-encoding TUB1 mRNA was considered as a potential cause of the cef1-13 cell cycle arrest because cef1-13 cells arrest uniformly at G₂/M with many hallmarks of a defective microtubule cytoskeleton. Consistent with this possibility, cef1-13 cells possess reduced levels of total TUB1 mRNA and α-tubulin protein. Removing the intron from TUB1 in cef1-13 cells boosts TUB1 mRNA and α-tubulin expression to near wild-type levels and restores microtubule stability in the cef1-13 mutant. As a result, cef1-13 tub1Δi cells progress through mitosis and their cell cycle arrest phenotype is alleviated. Removing the TUB1 intron from two other splicing mutants that arrest at G₂/M, prp17Δ and prp22-1 strains, permits nuclear division, but suppression of the cell cycle block is less efficient. Our data raise the possibility that although cell cycle arrest phenotypes in prp mutants can be explained by defects in pre-mRNA splicing, the transcript(s) whose inefficient splicing contributes to cell cycle arrest is likely to be prp mutant dependent.

We thank K. Gull, D. C. Kaiser, D. Kellogg, J. Patton, S. Reed, B. Rymond, and P. A. Weil for their generous gifts of strains, plasmids, and antibodies. We are grateful to D. McFarland and Michelle Thornsberry for flow cytometric analysis. Mary Morphew (Boulder Laboratory for 3D Fine Structure) kindly prepared specimens for electron micrograph analysis. J. Flick and all members of the Gould laboratory are acknowledged for providing useful discussions.

This work was supported by NIH grant GM47728 to K.L.G. Microarray work was supported by a grant from the W. M. Keck Foundation to the RNA Center at the University of California at Santa Cruz. C.G.B. was supported by NIH Medical Scientist Training Program grant GM07347. K.L.G. is an Associate Investigator of the Howard Hughes Medical Institute.