Functional conservation of the human homolog of the yeast pre-mRNA splicing factor Prp17p

Splicing of pre-mRNAs involves two sequential transesterification reactions commonly referred to as the first and second steps. In Saccharomyces cerevisiae, four proteins, Prp16p, Prp17p, Prp18p, and Slu7p are exclusively required for the second step of splicing. The human homologs of Prp16p, Prp17p, and Prp18p have been identified, and the human proteins hPrp16 and hPrp18 have been shown to be required for the second step of splicing in vitro. Here we provide further evidence for the functional conservation of the second step factors between yeast and humans. Human hPrp17, which is 35% identical to the S. cerevisiae protein, is able to partially rescue the temperature-sensitive phenotype in a yeast strain where PRP17 has been knocked out, suggesting that the human and yeast proteins are functionally conserved. Overexpression of hPrp17 in the knockout yeast strain partially rescues the splicing defect seen in vitro and in vivo. In HeLa cells, hPrp17 is highly concentrated in the nuclear speckles, as is SC35 and many other splicing factors, thus providing further support that this protein also functions as a splicing factor in humans.     

Cloning and characterization of a human DEAH-box RNA helicase, a functional homolog of fission yeast Cdc28/Prp8

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.     

Multiple distinct splicing enhancers in the protein-coding sequences of a constitutively spliced pre-mRNA

We have identified multiple distinct splicing enhancer elements within protein-coding sequences of the constitutively spliced human beta-globin pre-mRNA. Each of these highly conserved sequences is sufficient to activate the splicing of a heterologous enhancer-dependent pre-mRNA. One of these enhancers is activated by and binds to the SR protein SC35, whereas at least two others are activated by the SR protein SF2/ASF. A single base mutation within another enhancer element inactivates the enhancer but does not change the encoded amino acid. Thus, overlapping protein coding and RNA recognition elements may be coselected during evolution. These studies provide the first direct evidence that SR protein-specific splicing enhancers are located within the coding regions of constitutively spliced pre-mRNAs. We propose that these enhancers function as multisite splicing enhancers to specify 3' splice-site selection.     

The DEAH-box splicing factor Prp16 unwinds RNA duplexes in vitro

BACKGROUND: During pre-mRNA splicing, dynamic rearrangement of RNA secondary structure within the spliceosome is crucial for intron recognition and formation of the catalytic core. Splicing factors belonging to the DExD/DExH-box family of RNA-dependent ATPases are thought to have a central role in directing these rearrangements by unwinding RNA helices. Proof of this hypothesis has, however, been conspicuously lacking. RESULTS: Prp16 is a DEAH-box protein that functions in the second step of splicing in vitro. Using various RNA duplexes as substrate, we have shown that Prp16 has an ATP-dependent RNA unwinding activity. This activity is independent of sequence in either the single-stranded or duplexed regions of the RNA substrate. A mutation (prp16-1) near the ATP-binding motif of Prp16 inhibits both the RNA-dependent ATPase activity and the ATP-dependent RNA unwinding activity. CONCLUSIONS: Our findings provide strong biochemical evidence that Prp16 can disrupt a duplexed RNA structure on the spliceosome. Because the purified protein lacks sequence specificity in unwinding RNA duplexes, targeting of the unwinding activity of Prp16 in the spliceosome is likely to be determined by other interacting protein factors. The demonstration of unwinding activity will also help our understanding of how the fidelity of branchpoint recognition is controlled by Prp16.     

The budding yeast U5 snRNP Prp8 is a highly conserved protein which links RNA splicing with cell cycle progression

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.     

Efficient initiation of S-phase in yeast requires Cdc40p, a protein involved in pre-mRNA splicing

The S. cerevisiae CDC40 gene was originally identified as a cell-division-specific gene that is essential only at elevated temperatures. Cells carrying mutations in this gene arrest with a large bud and a single nucleus with duplicated DNA content. Cdc40p is also required for spindle establishment or maintenance. Sequence analysis reveals that CDC40 is identical to PRP17, a gene involved in pre-mRNA splicing. In this paper, we show that Cdc40p is required at all temperatures for efficient entry into S-phase and that cell cycle arrest associated with cdc40 mutations is independent of all the known checkpoint mechanisms. Using immunofluorescence, we show that Cdc40p is localized to the nuclear membrane, weakly associated with the nuclear pore. Our results point to a link between cell cycle progression, pre-mRNA splicing, and mRNA export.     

Cloning and characterization of PSF, a novel pre-mRNA splicing factor

Previously, we characterized cDNAs encoding polypyrimidine tract-binding protein (PTB) and showed that a complex between PTB and a 100-kD protein was necessary for pre-mRNA splicing. In this paper we have used two different in vitro-binding assays to confirm and extend the interaction between these two proteins. Peptide sequence information was used to clone and sequence cDNAs encoding alternatively spliced forms of the 100-kD protein. It contains two consensus RNA-binding domains and an unusual amino terminus rich in proline and glutamine residues. The protein is highly basic and migrates anomalously on SDS gels. Owing to its interaction with PTB and its role in pre-mRNA splicing, we have termed the 100-kD protein PTB-associated splicing factor (PSF). The RNA-binding properties of PSF are apparently identical to those of PTB. Both proteins, together and independently, bind the polypyrimidine tract of mammalian introns. Biochemical complementation, antibody inhibition, and immunodepletion experiments demonstrate that PSF is an essential pre-mRNA splicing factor required early in spliceosome formation. Bacterially synthesized PSF is able to complement immunodepleted extracts and restore splicing activity. Despite association with PSF, complementary experiments with antibodies against PTB do not suggest an essential role for PTB in pre-mRNA splicing.     

Evidence for a role for galectin-1 in pre-mRNA splicing

Galectins are a family of beta-galactoside-binding proteins that contain characteristic amino acid sequences in the carbohydrate recognition domain (CRD) of the polypeptide. The polypeptide of galectin-1 contains a single domain, the CRD. The polypeptide of galectin-3 has two domains, a carboxyl-terminal CRD fused onto a proline- and glycine-rich amino-terminal domain. In previous studies, we showed that galectin-3 is a required factor in the splicing of nuclear pre-mRNA, assayed in a cell-free system. We now document that (i) nuclear extracts derived from HeLa cells contain both galectins-1 and -3; (ii) depletion of both galectins from the nuclear extract either by lactose affinity adsorption or by double-antibody adsorption results in a concomitant loss of splicing activity; (iii) depletion of either galectin-1 or galectin-3 by specific antibody adsorption fails to remove all of the splicing activity, and the residual splicing activity is still saccharide inhibitable; (iv) either galectin-1 or galectin-3 alone is sufficient to reconstitute, at least partially, the splicing activity of nuclear extracts depleted of both galectins; and (v) although the carbohydrate recognition domain of galectin-3 (or galectin-1) is sufficient to restore splicing activity to a galectin-depleted nuclear extract, the concentration required for reconstitution is greater than that of the full-length galectin-3 polypeptide. Consistent with these functional results, double-immunofluorescence analyses show that within the nucleus, galectin-3 colocalizes with the speckled structures observed with splicing factor SC35. Similar results are also obtained with galectin-1, although in this case, there are areas of galectin-1 devoid of SC35 and vice versa. Thus, nuclear galectins exhibit functional redundancy in their splicing activity and partition, at least partially, in the nucleoplasm with another known splicing factor.     

Coinfection of a fungal pathogen by two distinct double-stranded RNA viruses

Unsegmented double-stranded (ds)RNA viruses belonging to the family Totiviridae persistently infect protozoa and fungi. In this study, two totiviruses were found to coinfect the filamentous fungus Sphaeropsis sapinea, a well known pathogen of pines. Isometric, virus-like particles approximately 35 nm in diameter were isolated from extracts of this fungus. The nucleotide sequences of the genomes of the two S. sapinea RNA viruses named SsRV1 and SsRV2 were established. The linear genomes of 5163 and 5202 bp, respectively, are identically organized with two large, overlapping ORFs. The 5' located ORF1 probably encodes the coat protein, whereas the gene product of ORF2 shows the typical features of RNA-dependent RNA polymerases. The absence of a pseudoknot and a slippery site at the overlapping region between ORF1 and ORF2, as well as the shortness of that region, leads us to suggest that the translation of ORF2 of both viruses is internally initiated. The mode of translation and the genomic organization are similar to those of Helminthosporium victoriae 190S virus (Hv190SV; Huang, S., and Ghabrial, S. A. (1996). Proc. Natl. Acad. Sci. USA 93, 12541-12546). Hv190SV thus appears to be closely related to the SsRVs. Interestingly, based on amino acid sequence homology SsRV1 is more closely related to Hv190SV than to SsRV2.     

Dbp5p, a cytosolic RNA helicase, is required for poly(A)+ RNA export

The DBP5 gene encodes a putative RNA helicase of unknown function in the yeast Saccharomyces cerevisiae. It is shown here that Dbp5p is an ATP-dependent RNA helicase required for polyadenylated [poly(A)+] RNA export. Surprisingly, Dbp5p is present predominantly, if not exclusively, in the cytoplasm, and is highly enriched around the nuclear envelope. This observation raises the possibility that Dbp5p may play a role in unloading or remodeling messenger RNA particles (mRNPs) upon arrival in the cytoplasm and in coupling mRNP export and translation. The functions of Dbp5p are likely to be conserved, since its potential homologues can be found in a variety of eukaryotic cells.     

Yrb2p is a nuclear protein that interacts with Prp20p, a yeast Rcc1 homologue

A conserved family of Ran binding proteins (RBPs) has been defined by their ability to bind to the Ran GTPase and the presence of a common region of approximately 100 amino acids (the Ran binding domain). The yeast Saccharomyces cerevisiae genome predicts only three proteins with canonical Ran binding domains. Mutation of one of these, YRB1, results in defects in transport of macromolecules across the nuclear envelope (Schlenstedt, G., Wong, D. H., Koepp, D. M., and Silver, P. A. (1995) EMBO J. 14, 5367-5378). The second one, encoded by YRB2, is a 327-amino acid protein with a Ran binding domain at its C terminus and an internal cluster of FXFG and FG repeats conserved in nucleoporins. Yrb2p is located inside the nucleus, and this localization relies on the N terminus. Results of both genetic and biochemical analyses show interactions of Yrb2p with the Ran nucleotide exchanger Prp20p/Rcc1. Yrb2p binding to Gsp1p (yeast Ran) as well as to a novel 150-kDa GTP-binding protein is also detected. The Ran binding domain of Yrb2p is essential for function and for its association with Prp20p and the GTP-binding proteins. Taken together, we suggest that Yrb2p may play a role in the Ran GTPase cycle distinct from nuclear transport.     

The PRP31 gene encodes a novel protein required for pre-mRNA splicing in Saccharomyces cerevisiae

The pre-mRNA splicing factor Prp31p was identified in a screen of temperature-sensitive yeast strains for those exhibiting a splicing defect upon shift to the non- permissive temperature. The wild-type PRP31 gene was cloned and shown to be essential for cell viability. The PRP31 gene is predicted to encode a 60 kDa polypeptide. No similarities with other known splicing factors or motifs indicative of protein-protein or RNA-protein interaction domains are discernible in the predicted amino acid sequence. A PRP31 allele bearing a triple repeat of the hemagglutinin epitope has been generated. The tagged protein is functional in vivo and a single polypeptide species of the predicted size was detected by Western analysis with proteins from yeast cell extracts. Functional Prp31p is required for the processing of pre-mRNA species both in vivo and in vitro, indicating that the protein is directly involved in the splicing pathway.     

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

Smad6 and Smad7 function as intracellular antagonists in transforming growth factor-beta (TGF-beta) signaling. Here we report the isolation of human Smad6, which is closely related to Smad7. Smad6 and Smad7 mRNAs were differentially expressed in lung cancer cell lines and were rapidly and directly induced by TGF-beta1, activin and bone morphogenetic protein-7. Cross-talk between TGF-beta and other signaling pathways was demonstrated by the finding that epidermal growth factor (EGF) induced the expression of inhibitory SMAD mRNA. Moreover, whereas the phorbol ester PMA alone had no effect, it potentiated the TGF-beta1-induced expression of Smad7 mRNA. Ectopic expression of anti-sense Smad7 RNA was found to increase the effect of TGF-beta1, supporting its role as a negative regulator in TGF-beta signaling. Thus, expression of inhibitory Smads is induced by multiple stimuli, including the various TGF-beta family members, whose action they antagonize.     

Glucose repression on RIM1, a gene encoding a mitochondrial single-stranded DNA-binding protein, in Saccharomyces cerevisiae: a possible regulation at pre-mRNA splicing

The mitochondrial single-stranded DNA-binding protein (SSB) encoded by anuclear gene, RIM1, is a homolog of Escherichia coli SSB. The addition of glucose decreased the amount of RIM1-mRNA in cells growing in a glycerol medium, but increased the amount of the immature RIM1-mRNA. The changes in the amounts of both mature and immature RIM1-mRNAs were dependent on SRN1/REG1/HEX2, a gene relating to pre-mRNA-splicing and glucose repression. These observations suggest that the expression of the mitochondrial SSB is regulated, at least in part, by pre-mRNA splicing under the control of glucose repression.     

Synthetic lethality of yeast slt mutations with U2 small nuclear RNA mutations suggests functional interactions between U2 and U5 snRNPs that are important for both steps of pre-mRNA splicing

A genetic screen was devised to identify Saccharomyces cerevisiae splicing factors that are important for the function of the 5' end of U2 snRNA. Six slt (stands for synthetic lethality with U2) mutants were isolated on the basis of synthetic lethality with a U2 snRNA mutation that perturbs the U2-U6 snRNA helix II interaction. SLT11 encodes a new splicing factor and SLT22 encodes a new RNA-dependent ATPase RNA helicase (D. Xu, S. Nouraini, D. Field, S. J. Tang, and J. D. Friesen, Nature 381:709-713, 1996). The remaining four slt mutations are new alleles of previously identified splicing genes: slt15, previously identified as prp17 (slt15/prp17-100), slt16/smd3-1, slt17/slu7-100, and slt21/prp8-21. slt11-1 and slt22-1 are synthetically lethal with mutations in the 3' end of U6 snRNA, a region that affects U2-U6 snRNA helix II; however, slt17/slu7-100 and slt21/prp8-21 are not. This difference suggests that the latter two factors are unlikely to be involved in interactions with U2-U6 snRNA helix II but rather are specific to interactions with U2 snRNA. Pairwise synthetic lethality was observed among slt11-1 (which affects the first step of splicing) and several second-step factors, including slt15/prp17-100, slt17/slu7-100, and prp16-1. Mutations in loop 1 of U5 snRNA, a region that is implicated in the alignment of the two exons, are synthetically lethal with slu4/prp17-2 and slu7-1 (D. Frank, B. Patterson, and C. Guthrie, Mol. Cell. Biol. 12:5179-5205, 1992), as well as with slt11-1, slt15/prp17-100, slt17/slu7-100, and slt21/prp8-21. These same U5 snRNA mutations also interact genetically with certain U2 snRNA mutations that lie in the helix I and helix II regions of the U2-U6 snRNA structure. Our results suggest interactions among U2 snRNA, U5 snRNA, and Slt protein factors that may be responsible for coupling and coordination of the two reactions of pre-mRNA splicing.     

The two isoenzymes for yeast NAD+-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation

The two homologous genes GPD1 and GPD2 encode the isoenzymes of NAD-dependent glycerol 3-phosphate dehydrogenase in the yeast Saccharomyces cerevisiae. Previous studies showed that GPD1 plays a role in osmoadaptation since its expression is induced by osmotic stress and gpd1 delta mutants are osmosensitive. Here we report that GPD2 has an entirely different physiological role. Expression of GPD2 is not affected by changes in external osmolarity, but is stimulated by anoxic conditions. Mutants lacking GPD2 show poor growth under anaerobic conditions. Mutants deleted for both GPD1 and GPD2 do not produce detectable glycerol, are highly osmosensitive and fail to grow under anoxic conditions. This growth inhibition, which is accompanied by a strong intracellular accumulation of NADH, is relieved by external addition of acetaldehyde, an effective oxidizer of NADH. Thus, glycerol formation is strictly required as a redox sink for excess cytosolic NADH during anaerobic metabolism. The anaerobic induction of GPD2 is independent of the HOG pathway which controls the osmotic induction of GPD1. Expression of GPD2 is also unaffected by ROX1 and ROX3, encoding putative regulators of hypoxic and stress-controlled gene expression. In addition, GPD2 is induced under aerobic conditions by the addition of bisulfite which causes NADH accumulation by inhibiting the final, reductive step in ethanol fermentation and this induction is reversed by addition of acetaldehyde. We conclude that expression of GPD2 is controlled by a novel, oxygen-independent, signalling pathway which is required to regulate metabolism under anoxic conditions.     

KUZ, a conserved metalloprotease-disintegrin protein with two roles in Drosophila neurogenesis

During neurogenesis in Drosophila both neurons and nonneuronal cells are produced from a population of initially equivalent cells. The kuzbanian (kuz) gene described here is essential for the partitioning of neural and nonneuronal cells during development of both the central and peripheral nervous systems in Drosophila. Mosaic analyses indicated that kuz is required for cells to receive signals inhibiting the neural fate. These analyses further revealed that the development of a neuron requires a kuz-mediated positive signal from neighboring cells. The kuz gene encodes a metalloprotease-disintegrin protein with a highly conserved bovine homolog, raising the possibility that kuz homologs may act in similar processes during mammalian neurogenesis.