Sen1p in Saccharomyces cerevisiae is a Type I DNA/RNA helicase. Mutations in the helicase domain perturb accumulation of diverse RNA classes, and Sen1p has been implicated in 3' end formation of non-coding RNAs. Using a combination of global and candidate-specific two hybrid screens, eight proteins were identified that interact with Sen1p. Interactions with three of the proteins were analyzed further: Rpo21p(Rpb1p), a subunit of RNA polymerase II, Rad2p, a deoxyribonuclease required in DNA repair, and Rnt1p (RNase III), an endoribonuclease required for RNA maturation. For all three interactions, the two-hybrid results were confirmed by co-immunoprecipitation experiments. Genetic tests designed to assess the biological significance of the interactions indicate that Sen1p plays functionally significant roles in transcription and transcription-coupled DNA repair. To investigate the potential role of Sen1p in RNA processing and to assess the functional significance of the Sen1p/Rnt1p interaction, we examined U5 snRNA biogenesis. We provide evidence that Sen1p functions in concert with Rnt1p and the exosome at a late step in 3' end formation of one of the two mature forms of U5 snRNA but not the other. The protein-protein and protein-RNA interactions reported here suggest that the DNA/RNA helicase activity of Sen1p is utilized for several different purposes in multiple gene expression pathways.
A single base change in the helicase superfamily 1 domain of the yeast Saccharomyces cerevisiae SEN1 gene results in a heat-sensitive mutation that alters the cellular abundance of many RNA species. We compared the relative amounts of RNAs between cells that are wild-type and mutant after temperature-shift. In the mutant several RNAs were found to either decrease or increase in abundance. The affected RNAs include tRNAs, rRNAs and small nuclear and nucleolar RNAs. Many of the affected RNAs have been positively identified and include end-matured precursor tRNAs and the small nuclear and nucleolar RNAs U5 and snR40 and snR45. Several small nucleolar RNAs co-immunoprecipitate with Sen1 but differentially associate with the wild-type and mutant protein. Its inactivation also impairs precursor rRNA maturation, resulting in increased accumulation of 35S and 6S precursor rRNAs and reduced levels of 20S, 23S and 27S rRNA processing intermediates. Thus, Sen1 is required for the biosynthesis of various functionally distinct classes of nuclear RNAs. We propose that Sen1 is an RNA helicase acting on a wide range of RNA classes. Its effects on the targeted RNAs in turn enable ribonuclease activity.
A Saccharomyces cerevisiae homolog to Drosophila melanogaster and mouse Tcp-1 encoding taiDless complex polypeptide 1 (TCP1) has been identified, sequenced, and mapped. The mouse t complex and many of its unusual properties have attracted the attention of geneticists for nearly 60 years (11,12,20,60). t alleles were first discovered by their interaction with a dominant T-locus mutation to produce a tailless phenotype in double heterozygous T/t animals. Heterozygous T/+ animals have a short tail, and homozygous T/T animals are embryonic lethals. Homozygous tx/tY males are sterile (37, 38), and heterozygous +/t animals have tails of normal length and are visually indistinguishable from normal wild-type +/+ animals. Compared with wild-type laboratory chromosome 17, the principal variation in t chromosomes consists of at least four inversions of about 1% of the mouse genome (30 Mbs of DNA) (21, 23). The t complex is the name given to the rearranged region. It contains about 100 genes (39), including several genes expressed in the testes whose products show t-allele-specific alterations.t chromosomes confer a number of effects, including disturbances of embryonic development, alterations of sperm differentiation and function, and transmission ratio distortion. While transmission of the t complex through females is normal, heterozygous t/+ males transmit the t-bearing chromosome to their progeny in excess of the Mendelian expectation. Genetic analysis of transmission ratio distortion suggests that it depends on the action of up to four distorter loci (Tcd-J to Tcd4) as well as a responder locus (Tcr) (2, 36-38, 56, 63). The molecular basis of transmission ratio distortion remains unknown.A number of candidate genes for the distorter and responder loci, including Tcp-1, have been isolated from the t complex (75). Tailless complex polypeptide 1 (TCP-1), a protein of 57 kDa, is expressed in large amounts during * Corresponding author.
Previously, we showed that the yeast Saccharomyces cerevisiae cold-sensitive mutation tcpl-I confers growth arrest concomitant with cytoskeletal disorganization and disruption of microtubule-mediated processes. We have identified two new recessive mutations, tcpl-2 and tcpl-3, that confer heat-and cold-sensitive growth. Cells carrying tcpl alleles were analyzed after exposure to the appropriate restrictive temperatures by cell viability tests, differential contrast microscopy, fluorescent, and immunofluorescent microscopy of DNA, tubulin, and actin and by determining the DNA content per cell. All three mutations conferred unique phenotypes indicative of cytoskeletal dysfunction. A causal relationship between loss of Tcplp function and the development of cytoskeletal abnormalities was established by double mutant analyses. Novel phenotypes indicative of allele-specific genetic interactions were observed when tcpl-l was combined in the same strain with tubl-1, tub2-402, actl-1, and actl-4, but not with other tubulin or actin mutations or with mutations in other genes affecting the cytoskeleton. Also, overproduction of wild-type Tcplp partially suppressed growth defects conferred by actl-l and actl-4. Furthermore, Tcplp was localized to the cytoplasm and the cell cortex. Based on our results, we propose that Tcplp is required for normal development and function of actin and microtubules either through direct or indirect interaction with the major cytoskeletal components. INTRODUCTIONMouse TCP1, which codes for Tcplp (tailless complex polypeptide 1), is abundantly expressed during spermiogenesis. The TCP1 gene is located on chromosome 17 in a region called the t complex. This region is associated with unusual genetic properties and has been under study for more than 60 years. Recessive t-alleles were originally discovered by their interaction with a dominant T locus mutation to produce a tailless phenotype in double mutant animals (Dobrovalskaia-Zawadskaia, 1927;Chesley, 1932;Gluecksohn-Waelsch, 1989).One of the effects conferred by the t-chromosomes is transmission ratio distortion (TRD), which results in male-specific chromosome transmission in vast excess of Mendelian expectations. Mouse Tcplp has been implicated to play a role in TRD (Silver and Remis, 1987).Tcpl homologues, proteins of '60 kDa, have been identified in organisms ranging from archaebacteria to humans (Silver et al., 1979;Silver, 1981;Willison et al., 1986Willison et al., , 1987Ursic and Ganetzky, 1988;Ahmad and Gupta, 1990, Morita et al., 1991;Trent et al., 1991;Ursic and Culbertson, 1991;Mori et al., 1992). The yeast Saccharomyces cerevisiae TCP1 gene is essential for cell viability. Tcplp in yeast, Drosophila melanogaster, and mouse share between 61 and 72% amino acid sequence identity (Ursic and Ganetzky, 1988;Ursic and Culbertson, 1991), suggesting a primordial function for the protein.The intracellular localization of Tcplp has not been clearly established. The mouse homologue was suggested to be an extracellular matrix protein (Silver and ...
The SEN) gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SENI gene and characterized the SENI mRNA, the SEN) gene product, the temperature-sensitive seni-) mutation, and three SENI null alleles. The SENI gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SENI, we detect a protein corresponding to the predicted molecular weight of SEN1. The SENI protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SENI protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPFI gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive seni-) allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.
The Saccharomyces cerevisiae SEN1 gene codes for a nuclear, ATP-dependent helicase which is embedded in a complex network of protein-protein interactions. Pleiotropic phenotypes of mutations in SEN1 suggest that Sen1 functions in many nuclear processes, including transcription termination, DNA repair, and RNA processing. Sen1, along with termination factors Nrd1 and Nab3, is required for the termination of noncoding RNA transcripts, but Sen1 is associated during transcription with coding and noncoding genes. Sen1 and Nrd1 both interact directly with Nab3, as well as with the C-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II. It has been proposed that Sen1, Nab3, and Nrd1 form a complex that associates with Rpb1 through an interaction between Nrd1 and the Ser 5 -phosphorylated (Ser 5 -P) CTD. To further study the relationship between the termination factors and Rpb1, we used two-hybrid analysis and immunoprecipitation to characterize sen1-R302W, a mutation that impairs an interaction between Sen1 and the Ser 2 -phosphorylated CTD. Chromatin immunoprecipitation indicates that the impairment of the interaction between Sen1 and Ser 2 -P causes the reduced occupancy of mutant Sen1 across the entire length of noncoding genes. For protein-coding genes, mutant Sen1 occupancy is reduced early and late in transcription but is similar to that of the wild type across most of the coding region. The combined data suggest a handoff model in which proteins differentially transfer from the Ser 5 -to the Ser 2 -phosphorylated CTD to promote the termination of noncoding transcripts or other cotranscriptional events for protein-coding genes. In eukaryotes, protein-coding genes and some noncoding genes are transcribed by RNA polymerase II (Pol II). Transcription occurs in three highly ordered stages: initiation, elongation, and termination. Order is maintained in part through the complex interplay between factors that bind to the C-terminal domain (CTD) of Rpb1, the largest subunit of Pol II (21, 64). The CTD is composed of heptad repeats with the consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 (12). The number of repeats varies in different organisms. In the yeast Saccharomyces cerevisiae, the CTD is composed of 26 heptad repeats, whereas there are 52 repeats in the CTD in humans (62).All three serine residues in the heptad repeats of the CTD are dynamically phosphorylated and dephosphorylated during the transcription cycle. The pattern of the phosphorylation of the CTD coordinates the events of the transcription cycle by serving as a binding scaffold for proteins needed at each stage of transcription, with the phosphorylation state of the CTD determining which factors bind and in what order. The process has been likened to a code that orchestrates the ordered assembly of factors required for transcription and associated processes (6,10,13,41,43).During initiation, a hypophosphorylated Pol II is recruited to the promoter and the preinitiation complex is formed (38). Once 8 to 9 nucleotides have been transcrib...
The yeast Sen1 protein was discovered by virtue of its role in tRNA splicing in vitro. To help determine the role of Sen1 in vivo, we attempted to overexpress the protein in yeast cells. However, cells with a high-copy SEN1-bearing plasmid, although expressing elevated amounts of SEN1 mRNA, show little increase in the level of the encoded protein, indicating that a posttranscriptional mechanism limits SEN1 expression. This control depends on an amino-terminal element of Sen1. Using a genetic selection for mutants with increased expression of Sen1-derived fusion proteins, we identified mutations in a novel gene, designated SEN3. SEN3 is essential and encodes a 945-residue protein with sequence similarity to a subunit of an activator of the 20S proteasome from bovine erythrocytes, called PA700. Earlier work indicated that the 20S proteasome associates with a multisubunit regulatory factor, resulting in a 26S proteasome complex that degrades substrates of the ubiquitin system. Mutant sen3-1 cells have severe defects in the degradation of such substrates and accumulate ubiquitin-protein conjugates. Most importantly, we show biochemically that Sen3 is a subunit of the 26S proteasome. These data provide evidence for the involvement of the 26S proteasome in the degradation of ubiquitinated proteins in vivo and for a close relationship between PA700 and the regulatory complexes within the 26S proteasome, and they directly demonstrate that Sen3 is a component of the yeast 26S proteasome.In the yeast Saccharomyces cerevisiae, mature tRNAs are produced from primary transcripts through a series of nuclear RNA-processing events that sometimes include the removal of an intron adjacent to the anticodon loop. Intron excision and tRNA ligation require the activities of three known enzymes: a heterotrimeric endonuclease that catalyzes both 5Ј and 3Ј cleavages, a monomeric tRNA ligase, and an NAD-dependent phosphotransferase (22) that removes a 2Ј-phosphate from the splice junction to produce mature tRNA (reviewed in reference 5). A number of genes that play a role in tRNA splicing have been identified. These include genes that encode or otherwise affect known enzyme activities, specifically SEN1 and SEN2, which are required for full endonuclease activity in vitro (40), and RLG1, which codes for the tRNA-splicing ligase (5).
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