Gotowa bibliografia na temat „Suppressor Screen for Cold Sensitive slu7-2”

AbstractThe highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.

The two large subunits of RNA polymerase II, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpb1-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpb1-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpb1-1 suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed.

The two large subunits of RNA polymerase II, RPB1 and RPB2, contain regions of extensive homology to the two large subunits of Escherichia coli RNA polymerase. These homologous regions may represent separate protein domains with unique functions. We investigated whether suppressor genetics could provide evidence for interactions between specific segments of RPB1 and RPB2 in Saccharomyces cerevisiae. A plasmid shuffle method was used to screen thoroughly for mutations in RPB2 that suppress a temperature-sensitive mutation, rpb1-1, which is located in region H of RPB1. All six RPB2 mutations that suppress rpb1-1 were clustered in region I of RPB2. The location of these mutations and the observation that they were allele specific for suppression of rpb1-1 suggests an interaction between region H of RPB1 and region I of RPB2. A similar experiment was done to isolate and map mutations in RPB1 that suppress a temperature-sensitive mutation, rpb2-2, which occurs in region I of RPB2. These suppressor mutations were not clustered in a particular region. Thus, fine structure suppressor genetics can provide evidence for interactions between specific segments of two proteins, but the results of this type of analysis can depend on the conditional mutation to be suppressed.

Abstract Microtubules in eukaryotic cells participate in a variety of nuclear and cytoplasmic structures, reflecting functional requirements and cell cycle position. We are studying the cellular regulation of microtubule assembly and organization in the yeast Saccharomyces cerevisiae. We screened for genes that when overexpressed suppress the growth phenotype of conditional mutants in alpha-tubulin that arrest with excess microtubules at the nonpermissive temperature (class 2 mutations). Here we describe one such suppressing element, called ATS1 (for Alpha Tubulin Suppressor). Overexpression of this gene rescues both the growth and microtubule phenotypes of all class 2 mutations, but not the cold-sensitive mutations that arrest with no microtubules (class 1 mutations). Deletion of ATS1 confers a modest slow growth phenotype which is slightly enhanced in strains containing both a deletion of ATS1 and a class 2 tub 1 mutation. The predicted ATS1 protein contains 333 amino acids and has considerable structural homology to the products of both the mammalian mitotic control gene RCC1 and the S. cerevisiae gene SRM1/PRP20. Overexpression of SRM1/PRP20 also suppresses class 2 mutants. The results suggest that this family of genes may participate in regulatory interactions between microtubules and the cell cycle.

Abstract Mutations in the Caenorhabditis elegans gene mec-8 were previously shown to cause defects in mechanosensation and in the structure and dye filling of certain chemosensory neurons. Using noncomplementation screens, we have identified eight new mec-8 alleles and a deficiency that uncovers the locus. Strong mec-8 mutants exhibit an incompletely penetrant cold-sensitive embryonic and larval arrest, which we have correlated with defects in the attachment of body muscle to the hypodermis and cuticle. Mutations in mec-8 strongly enhance the mutant phenotype of unc-52(viable) mutations; double mutants exhibit an unconditional arrest and paralysis at the twofold stage of embryonic elongation, a phenotype characteristic of lethal alleles of unc-52, a gene previously shown to encode a homolog of the core protein of heparan sulfate proteoglycan, found in basement membrane, and to be involved in the anchorage of myofilament lattice to the muscle cell membrane. We have identified and characterized four extragenic recessive suppressors of a mec-8; unc-52(viable) synthetic lethality. The suppressors, which define the genes smu-1 and smu-2, can weakly suppress all mec-8 mutant phenes. They also suppress the muscular dystrophy conferred by an unc-52(viable) mutation.

Abstract Abstract 4924 Background Human multiple myeloma (MM) is an incurable hematological malignancy at present, and screen for novel therapy remains an urgent need. The objective of this study was to assess the efficacy of natural compound EBSC-26 on multiple myeloma cells. Methods Inhibition of cell growth and proliferation of MM cell lines by compounds were assessed by WST-8 [2-(2-methoxy-4-nitrophenyl)-3-4-nitrophenyl)-5-(2,4- disulfophenyl)-2H-tetrazolium, monosodium salt] which allows sensitive colorimetric assays for the determination of the number of viable cells. Effects of compounds on cell cycle progression were analyzed by using flow cytometry. Apoptosis was evaluated by analysis of Annexin V. Microtubules were detected by immunofluorescence staining and confocal microscopy. Western blot and semi-quantitative/quantitative RT-PCR were performed to test protein/gene expression. Results EBSC-26 with a purity of up to 99.5%, was extracted from Centipeda minima (L.), a compositae plant used for the treatment of cold, nasal allergy, diarrhea, malaria, and asthma in China. We found that EBSC-26 suppressed proliferation/growth of U266, RPMI8226, dexamethasone-sensitive and resistant MM.1 cells, and induced apoptosis of these cells in a dose- and time-dependent manner. It synergized with Bortezomib and Doxorubicin in inhibition of MM cell proliferation. EBSC-26 overcame the protective effects of interleukin-6 and insulin-like growth factor-1 on multiple myeloma cells. It down-regulated interleukin-6-induced phosphorylation of STAT3 and insulin-like growth factor-1-induced phosphorylation of AKT. Moreover, EBSC-26 caused polymerization of microtubules, and induced G2/M arrest MM cells. Interestingly, an important G2/M-phase regulator, cyclin B1 was dramatically increased by EBSC-26 at protein level in a dose-dependent manner. EBSC-26 also decreased the phosphorylation of CDC2 at tyrosine 15. Conclusions These results suggest that EBSC-26 alone may have a potential in the treatment of multiple myeloma, and a combination of this agent with other compounds might provide further benefits. Disclosures No relevant conflicts of interest to declare.

The fission yeast genome with abundant multi-intronic transcripts, degenerate splice signals and presence of alternative splicing machinery is an attractive unicellular fungal model to investigate splice-site recognition and assemblymechanisms relevant to other fungal, worm, fly and higher eukaryotic genomes.Earlier work in the laboratory showed fission yeast SpSlu7 (homolog of budding yeast Slu7, Synergistic Lethal with U5-snRNA) has pre-catalytic intron context dependent splicing functions contrasting the predominant second step splicing role of budding yeast Slu7. Here we have investigated partners of SpSlu7 to discover its role splicing and other regulatory processes. Part I: Understanding SpSlu7 functional interactome by genetic and biochemical approaches We took up investigations todelineate the functional interactome of SpSlu7 that would mechanistically explain the pre-catalytic splicing arrest of spslu7-2 cells.Affinity purification of epitope tagged Slu7 associated complexes was donefollowed by mass spectrometry. The proteins associated with SpSlu7indicate its presence inboth pre-catalytic and second step spliceosomes. Genetic interaction assays were then carried out to validate and interpret these physical associations. Double mutants of slu7-2 with other splicing factor mutants, acting at distinct stages of the splicing pathway, were generated and studied. Taken together, we deduce an early pre-catalytic recruitment of SpSlu7 and a continued association afterthe first catalytic reaction. Further, in cells metabolically depleted of SpSlu7-2 the intron lariat intermediateRNA species was detected for intron 1 in transcript tfIID. This is a signature of poor second step splicing and thus demonstratesSpSlu7+functions improve second step splicing. Notably, other data from proteomic and genetic interaction studies showfunctional SpSlu7association with components of transcription, gene silencing and RNA decay machineries. Additionally the data uncovered a novel non-canonical Slu7function in meiotic RNA elimination in mitotically growing cells. In a parallel genetic screen for suppressorsof the cold sensitive slu7-2 therevertant UV10was obtained. Careful assessment of growth and cellular phenotypesconfirmed thatsuppression in UV10 was interactional and hinted an unanticipated effects on cellular pathways of mating and cell septation. Whole genome re-sequencing has been employed to identify candidate for the suppressor. Part II: Probing physiological relevance and mechanistic insights into SpSlu7 dependent fission yeast alternative splice events. The second part of the study probed possible physiological relevance and mechanistic insights of certain fission yeast exon skipping, intron retention and alternative splice site selection events and their dependence on SpSlu7. We identifiedstress and growth condition specific splice isoforms for certain transcripts.By assessment of the combinatorial effects of the intronic cis features, transcription elongation kinetics and splicing factor SpSlu7 activity, we deduced that suboptimal splice signals were the primary determinants of the low frequency usage of alternative splice sites. Additionally, we inferred use of certain alternative splice sites is co-transcriptional. Interestingly under conditions of mild thermal stress while SpSlu7 nuclear localization was unchanged, a distinct puncta like nuclear aggregation was noted foran interactor- branchpoint recognition protein U2AF59. These data underscorethe likelihood of stress-mediated remodelling of splicing factor complexes in fission yeast akin to higher eukaryotes.Overall, this study derived important insights on spliceosomal associations of splicing factor SpSlu7 and elucidated functions in splicing, meiotic RNA elimination, and stress-specific alternative splicing.