Isolation of an essential Schizosaccharomyces pombe gene, prp31+, that links splicing and meiosis

Bishop, D. Timothy; McDonald, W. Hayes; Gould, Kathleen L.; Forsburg, Susan L.

doi:10.1093/nar/28.11.2214

Cited by 12 publications

(5 citation statements)

References 33 publications

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“…For most genes, maximum inhibition of splicing was observed 12-14 hours following addition of thiamine suggesting that depletion of Ini1 may take several hours. Although the levels of accumulation of premRNA did not reach the levels detected in the prp2-1 mutant, they were clearly above background and are similar to data published for other splicing defective mutants (Bishop et al, 2000;Burns et al, 1999;Habara et al, 2001;Shimoseki and Shimoda, 2001;Urushiyama et al, 1996). The prp2-1 mutant is unique in displaying a very strong inhibition of splicing at the restrictive temperature.…”

Section: Resultssupporting

confidence: 83%

A novel RING-finger-like protein Ini1 is essential for cell cycle progression in fission yeast

Oltra

Verde

Werner

et al. 2004

Journal of Cell Science

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IntroductionMany factors influence the steady state levels of cellular mRNA. These include regulation of the rate of transcription, the rate of mRNA turnover, and the efficiency of posttranscriptional processing of pre-mRNA to form mature functional mRNA. To identify protein factors that are involved in the differential regulation of connexin43 (cx43) gene expression in rat heart and uterus, a uterine cDNA expression library was screened for proteins that interact directly with the cx43 gene promoter. This screen identified a protein called Ini, which binds to a 38-nucleotide region of the cx43 promoter (Oltra et al., 2003;Oltra and Werner, 1998). This sequence had been shown previously to function as a cis-activator in the transcription of the cx43 gene (Chen et al., 1995).Analysis of the Ini gene sequence demonstrates that it is evolutionarily conserved, sharing >70% identity with proteins from a wide variety of organisms from yeast to human. To further elucidate the potential function of the Ini protein, we have cloned a homologous sequence from fission yeast (Schizosaccharomyces pombe), called ini1 + , and investigated its putative function by gene disruption experiments. Deletion of ini1 + from S. pombe was lethal, and loss of Ini1 resulted in a block to cell cycle progression. Cell cycle arrest required the activity of the Wee1 protein kinase, a negative regulator of mitosis, but not Rad3, a kinase required for the G2-M checkpoint control. Therefore, the DNA damage checkpoint is not involved in the cell cycle delay.Some of the characteristics of the ini1 + gene and the phenotype of the ∆ini1 mutant were reminiscent of a collection of cdc/prp genes that have been shown to be required for normal pre-mRNA splicing in yeast (Burns et al., 1999;Habara et al., 2001;Lundgren et al., 1996;Ohi et al., 2002;Potashkin et al., 1998;Potashkin et al., 1989;Urushiyama et al., 1996). At least three of these genes, cdc5, cdc28 and prp12 + are required for cell viability and cell cycle progression (Habara et al., 2001;Lundgren et al., 1996;Ohi et al., 1994). Furthermore, a Saccharomyces cerevisiae homologue of ini1 + is present in purified preparations of the yeast U2/U5/U6 snRNP complex, which is required for pre-mRNA splicing (K. Gould, personal communication). These findings suggest that S. pombe ini1 + is required for pre-mRNA splicing. Consistent with this hypothesis, we show that six of seven intron-containing genes are incompletely spliced in cells depleted of Ini1. Therefore, Ini1 may be required for general splicing of pre-mRNA, at least some of which encode proteins required for normal cell cycle progression. Materials and MethodsYeast strains and methods All yeast strains were derived from S. pombe 972 and 975. All growth conditions, and genetic manipulations were as previously described (Moreno et al., 1991). For flow cytometry analysis of DNA content, S. pombe cells were stained with propidium iodide and analyzed using a Becton Dickinson FACScan as previously described (Sazer and Sherwood, 1990). Cloning and de...

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Section: Resultssupporting

confidence: 83%

A novel RING-finger-like protein Ini1 is essential for cell cycle progression in fission yeast

Oltra

Verde

Werner

et al. 2004

Journal of Cell Science

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“…Database searches with the complete amino acid sequence of the human 61K protein revealed that it is a homolog of the Prp31p protein of Saccharomyces cerevisiae (25% identity, 60% similarity) and Schizosaccharomyces pombe (68% similarity). Both proteins have been shown to be essential splicing factors in yeast (Weidenhammer et al ., 1996; Bishop et al ., 2000). The regions of homology are found throughout the entire sequence and are not restricted to one particular domain (Figure 1; data not shown); thus, these proteins are potentially orthologs.…”

Section: Resultsmentioning

confidence: 99%

Protein 61K, encoded by a gene (PRPF31) linked to autosomal dominant retinitis pigmentosa, is required for U4/U6middle dotU5 tri-snRNP formation and pre-mRNA splicing

et al. 2002

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contributed equally to this workIn each round of nuclear pre-mRNA splicing, the U4/ U6´U5 tri-snRNP must be assembled from U4/U6 and U5 snRNPs, a reaction that is at present poorly understood. We have characterized a 61 kDa protein (61K) found in human U4/U6´U5 tri-snRNPs, which is homologous to yeast Prp31p, and show that it is required for this step. Immunodepletion of protein 61K from HeLa nuclear extracts inhibits tri-snRNP formation and subsequent spliceosome assembly and pre-mRNA splicing. Signi®cantly, complementation with recombinant 61K protein restores each of these steps. Protein 61K is operationally de®ned as U4/U6 snRNP-speci®c as it remains bound to this particle at salt concentrations where the tri-snRNP dissociates. However, as shown by two-hybrid analysis and biochemical assays, protein 61K also interacts speci®cally with the U5 snRNP-associated 102K protein, indicating that it physically tethers U4/U6 to the U5 snRNP to yield the trisnRNP. Interestingly, protein 61K is encoded by a gene (PRPF31) that has been shown to be linked to autosomal dominant retinitis pigmentosa. Thus, our studies suggest that disruptions in tri-snRNP formation and function resulting from mutations in the 61K protein may contribute to the manifestation of this disease. Keywords: pre-mRNA splicing/retinitis pigmentosa/ snRNP protein function/spliceosome assembly/ U4/U6´U5 tri-snRNPs IntroductionThe splicing of pre-mRNA in the nucleus is catalyzed by a large ribonucleoprotein complex, the spliceosome (for reviews see Kra Èmer, 1996;Burge et al., 1999). The spliceosome consists of the pre-mRNA substrate and several small nuclear ribonucleoproteins (snRNPs), along with splicing factors not integrated into snRNPs. Each snRNP is a stable complex of a single RNA molecule (snRNA) and several proteins; some of these proteins are common to all snRNPs (the Sm proteins), while others are speci®c to a given snRNP ). The individual snRNPs interact during the splicing cycle in a highly dynamic manner. For example, at the start of the splicing cycle the U4 and U6 snRNPs are tightly associated by extensive RNA±RNA base-pairing, forming a single particle termed U4/U6. This complex associates in turn with U5 snRNP to yield the U4/U6´U5 tri-snRNP. The latter undergoes further rearrangements during the assembly of the catalytically active spliceosome. These include the partial or complete dissociation of U4 snRNP from the complex, the formation of new base-pairing between U6 and U2, and the interaction of U6 and U5 snRNAs with pre-mRNA sequences. After splicing, the spliceosome dissociates to the snRNP level, and the tri-snRNP must be re-assembled from U4, U5 and U6 snRNPs in order to take part in a new round of splicing (reviewed in Nilsen, 1998;Staley and Guthrie, 1998).Little is known about the interactions that mediate U4/U6 and U5 snRNP association. However, protein± protein interactions are believed to play a major part in tri-snRNP formation. Indeed, the tri-snRNP particle is particularly protein-rich. In humans it contains at least ...

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“…Connections between splicing and cell cycle progression have been known for several years. Mutations in genes coding for Prp proteins of S. cerevisiae or Prp homologues of S. pombe and causing cell cycle arrest were identified (Lundgreen et al , 1996; Bishop et al , 2000; Biggins et al , 2001). Recently, evidence is accumulating indicating a link between splicing and the cell cycle.…”

Section: Resultsmentioning

confidence: 99%

Proteins interacting with Lin1p, a putative link between chromosome segregation, mRNA splicing and DNA replication in Saccharomyces cerevisiae

Bialkowska

Kurlandzka

2002

Yeast

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Proteins involved in chromosome segregation during mitosis are likely to participate in other cell cycle-coordinated processes. Using a two-hybrid screen we identified a novel nuclear protein, Lin1, interacting with Irr1p/Scc3p, a component of the cohesin complex. The second round of two-hybrid assay with Lin1p as the bait resulted in the identification of six proteins: Prp8, Slx5, Siz2, Wss1, Rfc1 and YIL149w. These proteins have previously been shown to participate in mRNA splicing, DNA replication, chromosome condensation, chromatid separation and alternative cohesion. We propose that Lin1p may constitute a link among these processes.

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Isolation of an essential Schizosaccharomyces pombe gene, prp31+, that links splicing and meiosis

Cited by 12 publications

References 33 publications

A novel RING-finger-like protein Ini1 is essential for cell cycle progression in fission yeast

A novel RING-finger-like protein Ini1 is essential for cell cycle progression in fission yeast

Protein 61K, encoded by a gene (PRPF31) linked to autosomal dominant retinitis pigmentosa, is required for U4/U6middle dotU5 tri-snRNP formation and pre-mRNA splicing

Proteins interacting with Lin1p, a putative link between chromosome segregation, mRNA splicing and DNA replication in Saccharomyces cerevisiae

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