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.

No abstract

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 ...