Fission yeast Prp4p kinase regulates pre-mRNA splicing by phosphorylating a non-SR-splicing factor.

We provide evidence that Prp4p kinase activity is required for pre-mRNA splicing in vivo and show that loss of activity impairs G1-S and G2-M progression in the cell cycle. Prp4p interacts genetically with the non-SR (serine/arginine) splicing factors Prp1p and Prp5p. Bacterially produced Prp1p is phosphorylated by Prp4p in vitro. Prp4p and Prp1p also interact in the yeast two-hybrid system. In vivo labelling studies using a strain with a mutant allele of the prp4 gene in the genetic background indicate a change in phosphorylation of the Prp1p protein. These results are consistent with the notion that Prp4p kinase is involved in the control of the formation of active spliceosomes, targeting non-SR splicing factors.

Analysis of the splicing machinery in fission yeast: a comparison with budding yeast and mammals.

Based on genetic and bioinformatic analysis, 80 proteins from the newly sequenced Schizosaccharomyces pombe genome appear to be splicing factors. The fission yeast splicing factors were compared to those of Homo sapiens and Saccharomyces cerevisiae in order to determine the extent of conservation or divergence that has occurred over the billion years of evolution that separate these organisms. Our results indicate that many of the factors present in all three organisms have been well conserved throughout evolution. It is calculated that 38% of the fission yeast splicing factors are more similar to the human proteins than to the budding yeast proteins (>10% more similar or similar over a greater region). Many of the factors in this category are required for recognition of the 3' splice site. Ten fission yeast splicing factors, including putative regulatory factors, have human homologs, but no apparent budding yeast homologs based on sequence data alone. Many of the budding yeast factors that are absent in fission yeast are associated with the U1 and U4/U6.U5 snRNP. Collectively the data presented in this survey indicate that of the two yeasts, S.POMBE: contains a splicing machinery more closely reflecting the archetype of a spliceosome.

spp42, identified as a classical suppressor of prp4-73, which encodes a kinase involved in pre-mRNA splicing in fission yeast, is a homologue of the splicing factor Prp8p.

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.

Srp2, an SR protein family member of fission yeast: in vivo characterization of its modular domains.

We isolated srp2, a gene encoding a protein composed of two RNA binding domains (RBDs) at the N-terminus followed by an arginine-rich region that is flanked by two short SR (serine/arginine) elements. The RBDs contain the signatures RDADDA and SWQDLKD found in RBD1 and RBD2 of all typical metazoan SR proteins. srp2 is essential for growth. We have analyzed in vivo the role of the modular domains of Srp2 by testing specific mutations in a conditional strain for complementation. We found that RBD2 is essential for function and determines the specificity of RBD1 in Srp2. Replacement of the first RBD with RBD1 of Srp1 of fission yeast does not change this specificity. The two SR elements in the C-terminus of Srp2 are also essential for function in vivo. Cellular distribution analysis with green fluorescence protein fused to portions of Srp2 revealed that the SR elements are necessary to target Srp2 to the nucleus. Furthermore, overexpression of modular domains of Srp2 and Srp1 show different effects on pre-mRNA splicing activity of the tfIId gene. Taken together, these findings are consistent with the notion that the RBDs of these proteins may be involved in pre-mRNA recognition.

Cytoplasmic ribosomal protein genes of the fission yeast Schizosaccharomyces pombe display a unique promoter type: a suggestion for nomenclature of cytoplasmic ribosomal proteins in databases.

We identified 34 new ribosomal protein genes in the Schizosaccharomyces pombe database at the Sanger Centre coding for 30 different ribosomal proteins. All contain the Homol D-box in their promoter. We have shown that Homol D is, in this promoter type, the TATA-analogue. Many promoters contain the Homol E-box, which serves as a proximal activation sequence. Furthermore, comparative sequence analysis revealed a ribosomal protein gene encoding a protein which is the equivalent of the mammalian ribosomal protein L28. The budding yeast Saccharomyces cerevisiae has no L28 equivalent. Over the past 10 years we have isolated and characterized nine ribosomal protein (rp) genes from the fission yeast S.pombe . This endeavor yielded promoters which we have used to investigate the regulation of rp genes. Since eukaryotic ribosomal proteins are remarkably conserved and several rp genes of the budding yeast S.cerevisiae were sequenced in 1985, we probed DNA fragments encoding S.cerevisiae ribosomal proteins with genomic libraries of S.pombe . The deduced amino acid sequence of the different isolated rp genes of fission yeast share between 65 and 85% identical amino acids with their counterparts of budding yeast.

Identification and characterization of srp1, a gene of fission yeast encoding a RNA binding domain and a RS domain typical of SR splicing factors.

The SR protein family is involved in constitutive and regulated pre-mRNA splicing and has been found to be evolutionarily conserved in metazoan organisms. In contrast, the genome of the unicellular yeast Saccharomyces cerevisiae does not contain genes encoding typical SR proteins. The mammalian SR proteins consist of one or two characteristic RNA binding domains (RBD), containing the signature sequences RDAEDA and SWQDLKD respectively, and a RS (arginine/serine-rich) domain which gave the family its name. We have now cloned from the fission yeast Schizosaccharomyces pombe the gene srp1. This gene is the first yeast gene encoding a protein with typical features of mammalian SR protein family members. The gene is not essential for growth. We show that overexpression of the RNA binding domain inhibits pre-mRNA splicing and that the highly conserved sequence RDAEDA in the RBD is involved. Overexpression of Srp1 containing mutations in the RS domain also inhibits pre-mRNA splicing activity. Furthermore, we show that overexpression of Srp1 and overexpression of the mammalian SR splicing factor ASF/SF2 suppress the pre-mRNA splicing defect of the temperature-sensitive prp4-73 allele. prp4 encodes a protein kinase involved in pre-mRNA splicing. These findings are consistent with the notion that Srp1 plays a role in the splicing process.

Functional analysis of the fission yeast Prp4 protein kinase involved in pre-mRNA splicing and isolation of a putative mammalian homologue.

The prp4 gene of Schizosaccharomyces pombe encodes a protein kinase. A physiological substrate is not yet known. A mutational analysis of prp4 revealed that the protein consists of a short N-terminal domain, containing several essential motifs, which is followed by the kinase catalytic domain comprising the C-terminus of the protein. Overexpression of N-terminal mutations disturbs mitosis and produces elongated cells, Using a PCR approach, we isolated a putative homologue of Prp4 from human and mouse cells. The mammalian kinase domain is 53% identical to the kinase domain of Prp4. The short N-terminal domains share <20% identical amino acids, but contain conserved motifs. A fusion protein consisting of the N-terminal region from S. pombe followed by the mammalian kinase domain complements a temperature-sensitive prp4 mutation of S. pombe. Prp4 and the recombinant yeast/mouse protein kinase phosphorylate the human SR splicing factor ASF/SF2 in vitro in its RS domain.

Cloning and sequence analysis of a putative transcription factor (MTF1) gene from Mucor circinelloides.

We describe here the fortuitous cloning of a putative transcription factor gene (MTF1) from the dimorphic fungus Mucor circinelloides. Sequence analysis of MTF1 revealed an open reading frame (ORF) of 1059 nucleotides encoding a protein of M(r) 39601. The deduced amino acid sequence from the ORF imparts two glutamine-rich stretches which are homologous to a number of transcription factors characterized previously from various organisms. A Southern blot analysis of Mucor genomic DNA digested with different restriction endonucleases and probed with the 1.9 kb EcoR1 fragment of the putative transcription factor gene shows a single copy number of the the gene. Northern analysis during morphogenetic changes in Mucor suggested constitutive expression of the gene.

The tandem repeat AGGGTAGGGT is, in the fission yeast, a proximal activation sequence and activates basal transcription mediated by the sequence TGTGACTG.

Ribosomal protein (rp) genes in the fission yeast Schizosaccharomyces pombe display two highly conserved sequence elements in the promoter region. The molecular dissection of these promoters revealed that basal transcription is not based on a TATA element. The sequence which promotes basal transcription is the conserved sequence CAGTCACA or the inverted form TGTGACTG, called the homol D box. Upstream of the homol D box a tandem repeat AGGGTAGGGT or the inverted form ACCCTACCCT appears in some promoters, called homol E. This element functions in the proximal arrangement with homol D as an activation sequence. A compilation of homol D and homol E sequences identified in other S.pombe promoters revealed that several putative polymerase II and polymerase III promoters display a homol D box or the homol E/homol D arrangement.

Molecular mechanisms of TSD in reptiles: a search for the magic bullet.

Significant progress has been made in understanding mechanisms of genetic sex determination. The ZFY gene encodes a zinc finger protein but is not the primary signal in sex determination. The SRY gene is the testis determining gene in man, mouse, rabbit, and probably marsupial mouse and wallaby. Temperature dependent sex determination probably involves a modification of development of the indifferent gonad due to differential expression of one or more specific DNA sequences whose behavior is controlled by some temperature sensitive process or to differential action of a gene product such as a protein. There are ZFY and SRY-like genes in reptiles. We cloned and sequenced a portion of the ZFY gene (Zft) from snapping turtle (Chelydra serpentina) that is found in both sexes. We cloned and sequenced portions of SRY-like genes (Sra for SRY-related-autosomal) from snapping turtle. Similar genes are found in alligator (Alligator mississippiensis) and lizards. Cladistic analysis suggests that there are two or three major families of SRY-like genes in vertebrates in addition to sex specific SRY genes located on the Y chromosome of eutherian and marsupial mammals. When placed on a phylogenetic tree these data indicate that Sras were present in early tetrapods. Sequestering of the SRY gene on the Y chromosome probably happened only once and this may have been the defining moment that set the mammalian line of Therapsid reptiles apart from other reptilian groups.

Two genes encoding ribosomal protein L3 of Schizosaccharomyces pombe and their proximal promoter regions.

We have cloned and sequenced two genes, rpl3-1 and rpl3-2, encoding the ribosomal protein L3 of Schizosaccharomyces pombe. The two genes contain an open reading frame encoding 388 amino acids (aa) with a M(r) of 43,808. The aa sequences are identical, except at position 78, where Rpl3-1 displays a valine residue and Rpl3-2 contains isoleucine. The aa sequences show 75% identity to the RPL3 aa sequence from Saccharomyces cerevisiae. S1-nuclease protection analysis revealed that both genes are transcribed. The promoter sequences of the two rpl3 genes are significantly different, but both promoters contain the conserved homol-D element. Transcription starts between 40 and 50 nt downstream from this element.

Sequence analysis of the ZFY and Sox genes in the turtle, Chelydra serpentina.

We have sequenced regions of the ZFY and Sox genes in the turtle Chelydra serpentina, a reptile with temperature-dependent sex determination. The ZFY gene in mammals encodes a transcription factor with multiple zinc fingers that may be involved in spermatogenesis as well as other processes. The turtle homologue, Zft, is 92% identical to the ZFY gene at the nucleotide and amino acid levels in the region of zinc fingers 7-12. There are several Sox genes in the turtle that are only 57-70% identical at the nucleotide level and about 55% identical at the amino acid level to the human sex-determining SRY gene. However, the turtle Sox genes, termed TSox, have the conserved motif called the HMG-box (for high mobility group DNA-binding protein) that defines a probable DNA-binding region, and thus are in the same gene family as the Sox genes of other organisms from Drosophila to man. One TSox sequence is identical at the amino acid level to a sequence found in birds, and is 98% identical to a sequence encoded autosomally in mouse and in man. The extent of sequence conservation among the Sox genes suggests that some of their functions may be conserved. Phylogenetic analysis of available Sox sequences including SRY (Sry) sequences suggests that there was a high degree of divergence between any possible immediate common ancestor of the turtle Sox sequences and the SRY (Sry) sequences.

The fission yeast prp4+ gene involved in pre-mRNA splicing codes for a predicted serine/threonine kinase and is essential for growth.

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.

The CAGTCACA box in the fission yeast Schizosaccharomyces pombe functions like a TATA element and binds a novel factor.

Fourteen ribosomal protein genes from the fission yeast Schizosaccharomyces pombe contain a highly conserved sequence, CAGTCACA, in the proximal promoter. This sequence, which was also conserved in its location, was found where the TATA element usually resides. Deletion and point mutations in the CAGTCACA box reduced the expression of these genes to almost zero and caused aberrant transcriptional start sites. Insertions between this box and the original transcriptional start sites led to new start sites which were the same distance from the CAGTCACA box as the original start sites. The results presented provide evidence that this box, like a TATA sequence, is involved in basal expression and fixing the transcriptional start sites of these genes. Furthermore, the CAGTCACA sequence is the target of a binding protein which appears to be different from the TATA-binding protein.

The carboxypeptidase Y-encoding gene from Candida albicans and its transcription during yeast-to-hyphae conversion.

We have cloned and sequenced the gene (CPY1) encoding the carboxypeptidase Y (CPY) of Candida albicans. The gene contains an open reading frame comprising 542 amino acids (aa) with an M(r) of 61,104. The aa sequence shows 74% identity to the mature CPY aa sequence from Saccharomyces cerevisiae. The putative pre (signal) and pro sequences at the N terminus of the C. albicans protein, however, show significant divergence from the corresponding prepro sequence of the S. cerevisiae protein. Southern analysis of C. albicans genomic DNA suggested the presence of only one CPY-encoding gene. Northern analysis during yeast-to-hyphae conversion suggested that the CPY1 gene is transiently down-regulated on a transcriptional level during the early events of this developmental switch.

Architectural features of pre-mRNA introns in the fission yeast Schizosaccharomyces pombe.

The architectural features of 73 introns found in 36 genes of the fission yeast Schizosaccharomyces pombe have been compiled and tabulated. The introns from S. pombe can be grouped into two size classes. Intron features are discussed in comparison to intron features of Saccharomyces cerevisiae and other eukaryotes. The results indicate that S. pombe displays quite different architectural features than the budding yeast S. cerevisiae. However, particularly in the 3' region, S. pombe introns also appear to differ from mammalian introns.

prp4 from Schizosaccharomyces pombe, a mutant deficient in pre-mRNA splicing isolated using genes containing artificial introns.

We have generated a bank of temperature-sensitive (ts) Schizosaccharomyces pombe mutant strains. About 150 of these mutants were transformed with a ura4 gene containing an artificial intron. We screened these ts mutants for mutants deficient in splicing of the ura4 intron. With this approach three mutants were isolated which have a general defect in the splicing process. Two of these mutants fall into the prp1 complementation group and one defines a new complementation group, prp4.

Expression of the beta-glucuronidase gene under the control of the CaMV 35s promoter in Schizosaccharomyces pombe.

We have transformed Schizosaccharomyces pombe with the beta-glucuronidase (GUS) gene from Escherichia coli under the control of the plant cauliflower mosaic virus (CaMV) 35S promoter element. Efficient expression of GUS enzyme was observed. Moreover, transcription initiated at a unique site identical to that used in plant cells.