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 ␣-tubulin-encoding TUB1 mRNA was considered as a potential cause of the cef1-13 cell cycle arrest because cef1-13 cells arrest uniformly at G 2 /M with many hallmarks of a defective microtubule cytoskeleton. Consistent with this possibility, cef1-13 cells possess reduced levels of total TUB1 mRNA and ␣-tubulin protein. Removing the intron from TUB1 in cef1-13 cells boosts TUB1 mRNA and ␣-tubulin expression to near wild-type levels and restores microtubule stability in the cef1-13 mutant. As a result, cef1-13 tub1⌬i cells progress through mitosis and their cell cycle arrest phenotype is alleviated. Removing the TUB1 intron from two other splicing mutants that arrest at G 2 /M, prp17⌬ 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.Pre-mRNA splicing and cell cycle regulation have two distinct and apparently nonoverlapping functions for eukaryotic cells. In spite of this, a handful of genes in Saccharomyces cerevisiae and Schizosaccharomyces pombe have been identified in genetic screens for splicing factors (prp screens) and independently in screens for cell cycle regulators (cdc and related screens). These genes include S. cerevisiae PRP3 (also known as DBF5), PRP8 (also known as DBF3), PRP17 (also known as CDC40), and PRP22 and S. pombe prp8 ϩ (also known as cdc28 ϩ ) and prp2 ϩ (also known as mis11 ϩ ) (24,26,29,44,49,56,57). Furthermore, several prp mutants in S. pombe display morphologies consistent with defects in cell cycle progression (36, 54). Lastly, two proteins in S. pombe, Cdc5p and Dsk1p, both of which were identified genetically as potential cell cycle regulators, have since been implicated biochemically in premRNA splicing (30,50). In higher eukaryotes, splicing factors reorganize spatially throughout the cell cycle (19), and CDK2-cyclin E phosphorylates the U2 snRNP protein, SAP155 (43). Collectively, these data raise the possibility that pre-mRNA splicing and cell cycle control are functionally linked in vivo, although the molecular mechanisms underlying this connection remain vague.The conser...

PLRG1 Is an Essential Regulator of Cell Proliferation and Apoptosis during Vertebrate Development and Tissue Homeostasis

“…Previous mass spectrometry analyses of CDC5L-associated proteins have identified numerous spliceosomal components, including ASF/SF2, hnRNP-G, SAP145, and U2A, that are associated with PLRG1, and subsequent work has demonstrated a critical role for the PLRG1-CDC5L complex in the control of pre-mRNA splicing (2). Moreover, in yeast, the CDC5L homologue Prp46p regulates cell cycle progression via the control of tubulin splicing (11). Given the cytoplasmic relocalization of CDC5L in the absence of PLRG1, we next …”

“…Although the molecular mechanisms resulting in cell cycle defects as a consequence of the deletion of spliceosomal components have not been fully characterized, previous experiments in yeast demonstrated that defective pre-mRNA splicing directly blocks mitosis. Thus, the altered splicing of a single intron from the TUB1 ␣-tubulin gene caused by the CDC5 mutation results in the G 2 /M cell cycle defect in fission yeast (11). Moreover, besides the direct effect of the impaired splicing of critical cell cycle regulators, stalled splicing may lead to the rehybridization of nascent transcripts with the DNA template and the introduction of DNA breaks and genomic instability that ultimately leads to cell death (27).…”

Characterization of interactions among the Cef1p-Prp19p-associated splicing complex

“…The importance of these protein-protein interactions in vivo was confirmed by genetic analyses+ First, we tested whether overexpression of the various genes could suppress temperature-sensitive phenotypes of cef1-13 (Burns et al+, 2002), prp19-1 (Chen et al+, 1998), or snt309⌬ (Chen et al+, 1998) Second, we examined whether there were synthetic lethal interactions between cef1-13, prp19-1, and snt309⌬. The cef1-1 prp19-1 and the cef1-13 snt309⌬ double mutants were able to form colonies on medium selecting for a CEF1-expressing plasmid, but they were inviable on medium containing 5-FOA (Fig+ 4B,C)+ We conclude that cef1-13 is synthetically lethal with both prp19-1 and snt309⌬+ SNT309 was isolated in a synthetic lethal screen with prp19-4 (Chen et al+, 1998)+ Surprisingly these authors did not find that snt309⌬ and prp19-1 were synthetically lethal+ The ability of increased PRP19 expression to suppress snt309⌬ led us to reexamine this genetic interaction+ A CEN URA3-marked plasmid carrying a genomic clone of PRP19 was introduced into a MATa/a prp19-1/PRP19 snt309⌬/SNT309 double heterozygote prior to sporulation+ A haploid double mutant strain carrying the plasmid was isolated+ Although the prp19-1 snt309⌬ double mutant was able to form colonies on medium selecting for the PRP19-expressing plasmid, it was inviable on medium containing 5-FOA (Fig+ 4D)+ Thus, we conclude that prp19-1 and snt309⌬ are synthetically lethal+ In sum, these numerous genetic interactions support the importance of the direct physical associations described above+…”

Graded requirement for the spliceosome in cell cycle progression

“…17 Different snRNPs and accessory proteins are recruited or excluded at different steps of the splicing process, resulting in a very dynamic composition of the spliceosome machine and the generation of extensive rearrangements during different stages of splicing. 17,18 A connection between the spliceosome and cell cycle progression has been found in many organisms including budding yeast, [19][20][21][22][23][24] fission yeast, [25][26][27] Drosophila, 9,28 chicken, 29 mouse, 30 and human cells. 6,11,12,29,31,32 In human cells, depletion of different spliceosome components with siRNAs results in multiple cell cycle defects, with most siRNAs analyzed eliciting mitotic defects 6,11,12,31 although accumulation of cells in S phase 32 has also been observed.…”