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The purified yeast pre-mRNA splicing factor PRP2 is an RNA-dependent NTPase. 总被引:24,自引:7,他引:17
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Unlike autocatalyzed self-splicing reactions, nuclear pre-mRNA splicing requires transacting macromolecules and ATP. A protein encoded by the PRP2 gene of Saccharomyces cerevisiae is required, in conjunction with ATP, for the first cleavage-ligation reaction of pre-mRNA splicing. In this study, we have purified two forms of the PRP2 gene product with apparent molecular weights of 100 kDa and 92 kDa, from a yeast strain overproducing the protein. Both proteins were indistinguishable in their ability to complement extracts derived from a heat-sensitive prp2 mutant. Furthermore, we show that the PRP2 protein is capable of hydrolyzing nucleoside triphosphates in the presence of single-stranded RNAs such as poly(U). However, purified PRP2 by itself did not unwind double-stranded RNA substrates. The fact that an RNA-dependent NTPase activity is intrinsic to PRP2 may account for the ATP requirement in the first catalytic reaction of pre-mRNA splicing. 相似文献
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The fission yeast prp4+ gene involved in pre-mRNA splicing codes for a predicted serine/threonine kinase and is essential for growth. 总被引:5,自引:0,他引:5
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Only four prp (pre-mRNA processing) genes of the fission yeast Schizosaccharomyces pombe have been reported. We exploited yeast genetics and identified and isolated the prp4 gene. Sequence analysis revealed that the splicing factor encoded by this gene contains the signature sequences that define the serine/threonine protein kinase family. This is the first kinase gene identified whose product is involved in pre-mRNA splicing. The prp4 gene contains one intron in the kinase domain. Gene replacement studies provided evidence that this gene is essential for growth and is located on chromosome III. 相似文献
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Agata Jacewicz Beate Schwer Paul Smith Stewart Shuman 《Nucleic acids research》2014,42(20):12885-12898
Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5′ splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1–89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127–588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127–588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to the adenine nucleobase. The triphosphate moiety of AMPPNP•Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg2+•RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. Overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects. 相似文献
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The cell cycle control gene cdc2+ of fission yeast encodes a protein kinase potentially regulated by phosphorylation 总被引:75,自引:0,他引:75
The cdc2+ gene function has an important role in controlling the commitment of the fission yeast cell to the mitotic cycle and the timing of mitosis. We have raised antibodies against the cdc2+ protein using synthetic peptides and have demonstrated that it is a 34 kd phosphoprotein with protein kinase activity. The protein level and phosphorylation state remain unchanged during the mitotic cycle of rapidly growing cells. When cells cease to proliferate and arrest in G1 the protein becomes dephosphorylated and loses protein kinase activity. Exit from the mitotic cycle and entry into stationary phase may be controlled in part by modulation of the cdc2 protein kinase activity by changes in its phosphorylation state. 相似文献
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We have identified two classical extragenic suppressors, spp41 and spp42, of the temperature sensitive (ts) allele prp4-73. The prp4(+) gene of Schizosaccharomyces pombe encodes a protein kinase. Mutations in both suppressor genes suppress the growth and the pre-mRNA splicing defect of prp4-73(ts) at the restrictive temperature (36 degrees ). spp41 and spp42 are synthetically lethal with each other in the presence of prp4-73(ts), indicating a functional relationship between spp41 and spp42. The suppressor genes were mapped on the left arm of chromosome I proximal to the his6 gene. Based on our mapping data we isolated spp42 by screening PCR fragments for functional complementation of the prp4-73(ts) mutant at the restrictive temperature. spp42 encodes a large protein (p275), which is the homologue of Prp8p. This protein has been shown in budding yeast and mammalian cells to be a bona fide pre-mRNA splicing factor. Taken together with other recent genetic and biochemical data, our results suggest that Prp4 kinase plays an important role in the formation of catalytic spliceosomes. 相似文献
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Hub1/Ubl5 is a member of the family of ubiquitin-like proteins (UBLs). The tertiary structure of Hub1 is similar to that of ubiquitin; however, it differs from known modifiers in that there is no conserved glycine residue near the C terminus which, in ubiquitin and UBLs, is required for covalent modification of target proteins. Instead, there is a conserved dityrosine motif proximal to the terminal nonconserved amino acid. In S. cerevisiae, high molecular weight adducts can be formed in vivo from Hub1, but the structure of these adducts is not known, and they could be either covalent or noncovalent. The budding yeast HUB1 gene is not essential, but Delta hub1 mutants display defects in mating. Here, we report that fission yeast hub1 is an essential gene, whose loss results in cell cycle defects and inefficient pre-mRNA splicing. A screen for Hub1 interactors identified Snu66, a component of the U4/U6.U5 tri-snRNP splicing complex. Furthermore, overexpression of Snu66 suppresses the lethality of a hub1ts mutant. In cells lacking functional hub1, the nuclear localization of Snu66 is disrupted, suggesting that an important role for Hub1 is the correct subcellular targeting of Snu66, although our data suggest that Hub1 is likely to perform other roles in splicing as well. 相似文献
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Biochemical and genetic experiments have shown that the PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that plays a role during the second catalytic step of the splicing reaction. It was found recently that PRP17 is identical to the cell division cycle CDC40 gene. cdc40 mutants arrest at the restrictive temperature after the completion of DNA replication. Although the PRP17/CDC40 gene product is essential only at elevated temperatures, splicing intermediates accumulate in prp17 mutants even at the permissive temperature. In this report we describe extensive genetic interactions between PRP17/CDC40 and the PRP8 gene. PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and for both catalytic steps of the splicing reaction. We show that mutations in the PRP8 gene are able to suppress the temperature-sensitive growth phenotype and the splicing defect conferred by the absence of the Prp17 protein. In addition, these mutations are capable of suppressing certain alterations in the conserved PyAG trinucleotide at the 3' splice junction, as detected by an ACT1-CUP1 splicing reporter system. Moreover, other PRP8 alleles exhibit synthetic lethality with the absence of Prp17p and show a reduced ability to splice an intron bearing an altered 3' splice junction. On the basis of these findings, we propose a model for the mode of interaction between the Prp8 and Prp17 proteins during the second catalytic step of the splicing reaction. 相似文献
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Ukimori S Kawabata N Shimada H Imano R Takahashi K Yukawa M Tsuchiya E Ueno M 《Bioscience, biotechnology, and biochemistry》2012,76(2):264-269
In the fission yeast Schizosaccharomyces pombe, deletion of trt1(+) causes gradual telomere shortening, while deletion of pot1(+) causes rapid telomere loss. The double mutant between pot1 and RecQ helicase rqh1 is synthetically lethal. We found that the trt1 rqh1 double mutant was not synthetically lethal. The chromosome end fragments in both the trt1Δ rqh1Δ and the trt1Δ rqh1-hd (helicase dead) double mutants did not enter a pulsed-field electrophoresis gel. Both the trt1Δ rqh1Δ and the trt1Δ rqh1-hd double mutants were sensitive to the anti-microtubule drug thiabendazole. Moreover, the trt1Δ rqh1-hd double mutant displayed RPA foci on the chromosome bridge at high frequency in M phase cells. These phenotypes are very similar to that of the pot1Δ rqh1-hd double mutant, in which recombination intermediates accumulate at the chromosme ends in the M phase. These results suggest that the entangled chromosome ends, most likely recombination intermediates, are present in the M phase in the trt1Δ rqh1-hd double mutant. 相似文献
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Cloning and characterization of a human DEAH-box RNA helicase, a functional homolog of fission yeast Cdc28/Prp8. 总被引:3,自引:1,他引:2
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O Imamura K Saiki T Tani Y Ohshima M Sugawara Y Furuichi 《Nucleic acids research》1998,26(9):2063-2068
During the splicing process, spliceosomal snRNAs undergo numerous conformational rearrangements that appear to be catalyzed by proteins belonging to the DEAD/H-box superfamily of RNA helicases. We have cloned a new RNA helicase gene, designated DBP2 (DEAH-boxprotein), homologous to the Schizosaccaromyces pombe cdc28(+)/prp8(+) gene involved in pre-mRNA splicing and cell cycle progression. The full-length DBP2 contains 3400 nucleotides and codes for a protein of 1041 amino acids with a calculated mol. wt of 119 037 Da. Transfection experiments demonstrated that the GFP-DBP2 gene product, transiently expressed in HeLa cells, was localized in the nucleus. The DBP2 gene was mapped by FISH to the MHC region on human chromosome 6p21.3, a region where many malignant, genetic and autoimmune disease genes are linked. Because the expression of DBP2 gene in S.pombe prp8 mutant cells partially rescued the temperature-sensitive phenotype, we conclude that DBP2 is a functional human homolog of the fission yeast Cdc28/Prp8 protein. 相似文献
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The budding yeast U5 snRNP Prp8 is a highly conserved protein which links RNA splicing with cell cycle progression. 总被引:10,自引:3,他引:7
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The dbf3 mutation was originally obtained in a screen for DNA synthesis mutants with a cell cycle phenotype in the budding yeast Saccharomyces cerevisiae. We have now isolated the DBF3 gene and found it to be an essential gene with an ORF of 7239 nucleotides, potentially encoding a large protein of 268 kDa. We also obtained an allele-specific high copy number suppressor of the dbf3-1 allele, encoded by the known SSB1 gene, a member of the Hsp70 family of heat shock proteins. The sequence of the Dbf3 protein is 58% identical over 2300 amino acid residues to a predicted protein from Caenorhabditis elegans. Furthermore, partial sequences with 61% amino acid sequence identity were deduced from two files of human cDNA in the EST nucleotide database so that Dbf3 is a highly conserved protein. The nucleotide sequence of DBF3 turned out to be identical to the yeast gene PRP8, which encodes a U5 snRNP required for pre-mRNA splicing. This surprising result led us to further characterise the phenotype of dbf3 which confirmed its role in the cell cycle and showed it to function early, around the time of S phase. This data suggests a hitherto unexpected link between pre-mRNA splicing and the cell cycle. 相似文献
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Identification of p34 and p13, human homologs of the cell cycle regulators of fission yeast encoded by cdc2+ and suc1+ 总被引:41,自引:0,他引:41
cdc2+ and CDC28 play central roles in the cell division cycles of the widely divergent yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. The genes encode protein kinases that show 62% protein sequence identity and are capable of cross-complementation. Monoclonal antibodies were raised against p34cdc2, and a subset recognize p36cdc28. The cross-reacting antibodies detected a 34 kd homolog of the p34cdc2/p36CDC28, protein in HeLa cells. Human p34 was also recognized by an affinity-purified polyclonal anti-p34cdc2 serum. Peptide mapping of p34cdc2, p36CDC28, and human p34 revealed complete conservation of four tryptophan residues in the three proteins. p34 thus appears to be closely related to the two yeast proteins. In addition, a p34 immune complex showed protein kinase activity in vitro, and HeLa cell p34 interacts with p13, the human homolog of the suc1+ gene product of S. pombe. 相似文献
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suc1 is an essential gene involved in both the cell cycle and growth in fission yeast 总被引:18,自引:4,他引:14
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The gene suc1 encodes a product which suppresses certain temperature sensitive mutants of the cell cycle control gene cdc2 of Schizosaccharomyces pombe. Mutants in the suc1 gene or over-expression of its product leads to delays in mitotic and meiotic nuclear division. Deletion of the suc1 gene is lethal and generates some cells blocked in the cell cycle and others impaired in cellular growth. It is likely that the suc1 gene product binds and forms unstable complexes with the cdc2 protein kinase and with other proteins necessary for the cell cycle and cellular growth. suc1 may have a regulatory role in these processes. 相似文献
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The human U5 snRNP-specific 100-kD protein is an RS domain-containing, putative RNA helicase with significant homology to the yeast splicing factor Prp28p. 总被引:8,自引:4,他引:4
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Through UV-crosslinking experiments, we previously provided evidence suggesting that a U5 snRNP protein with a molecular weight in the 100-kDa range is an ATP-binding protein (Laggerbauer B, Lauber J, Lührmann R, 1996, Nucleic Acid Res 24:868-875). Separation of HeLa U5 snRNP proteins on 2D gels revealed multiple variants with apparent molecular masses of 100 kDa. Subsequent microsequencing of these variants led to the isolation of a cDNA encoding a protein with an N-terminal RS domain and a C-terminal domain that contains all of the conserved motifs characteristic of members of the DEAD-box family of RNA-stimulated ATPases and RNA helicases. Antibodies raised against cDNA-encoded 100-kDa protein specifically recognized native U5-100kD both on immunoblots and in purified HeLa U5 snRNPs or [U4/U6.U5] tri-snRNP complexes, confirming that the bona fide 100-kDa cDNA had been isolated. In vitro phosphorylation studies demonstrated that U5-100kD can serve as a substrate for both Clk/Sty and the U1 snRNP-associated kinase, and further suggested that the multiple U5-100kD variants observed on 2D gels represent differentially phosphorylated forms of the protein. A database homology search revealed a significant degree of homology (60% similarity, 37% identity) between the Saccharomyces cerevisiae splicing factor, Prp28p, which lacks an N-terminal RS domain, and the C-terminal domain of U5-100kD. Consistent with their designation as structural homologues, anti-Prp28 antibodies recognized specifically the human U5-100kD protein on immunoblots. Together with the DEXH-box U5-200kD protein (Lauber J et al., 1996, EMBO J 15:4001-4015), U5-100kD is the second example of a putative RNA helicase that is tightly associated with the U5 snRNP. Given the recent identification of the U5-116kD protein as a homologue of the ribosomal translocase EF-2 (Fabrizio P, Laggerbauer B, Lauber J, Lane WS, Lührmann R, 1997, EMBO J 16:4092-4106), at least three integral U5 snRNP proteins thus potentially facilitate conformational changes in the spliceosome during nuclear pre-mRNA splicing. 相似文献
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Shimada M Namikawa-Yamada C Nakanishi M Murakami H 《The Journal of biological chemistry》2005,280(38):32640-32648
Screening of cdc mutants of fission yeast for those whose cell cycle arrest is independent of the DNA damage checkpoint identified the RNA splicing-deficient cdc28 mutant. A search for mutants of cdc28 cells that enter mitosis with unspliced RNA resulted in the identification of an orb5 point mutant. The orb5+ gene, which encodes a catalytic subunit of casein kinase II, was found to be required for cell cycle arrest in other mutants with defective RNA metabolism but not for operation of the DNA replication or DNA damage checkpoints. Loss of function of wee1+ or rad24+ also suppressed the arrest of several splicing mutants. Overexpression of the major B-type cyclin Cdc13p induced cdc28 cells to enter mitosis. The abundance of Cdc13p was reduced, and the phosphorylation of Cdc2p on tyrosine 15 was maintained in splicing-defective cells. These results suggest that regulation of Cdc13p and Cdc2p is required for G2 arrest in splicing mutants. 相似文献