Project description:Puf3 is a RNA-binding protein, a member of the conserved Puf-protein family. Combining different functional genomics data, we have analyzed the role of Puf3 in post-transcriptional gene regulation in S. pombe. We present data on Puf3 interacting proteins and regulatory mRNA targets.
Project description:Expression profiles of polg mutant cells reveal that many genes encoding proteins involved in cell wall biogenesis and stress response are induced, suggesting that polg mutant cells attempt to maintain growth potential and undergo extensive oxidative metabolism. Conversely, many genes encoding proteins involved in ribosome biogenesis and respiration are repressed, indicating that cells depleted of mtDNA are adapted to grow slowly in absence of mitochondrial function. Keywords: Schizosaccharomyces pombe ployg mutant cells vs wild type cells.
Project description:Tristetraprolin is a vertebrate CCCH tandem zinc finger protein that can bind to and destabilize certain mRNAs containing AU-rich element binding sites. zfs1 is the single gene in the fission yeast, Schizosaccharomyces pombe, that encodes a protein containing the critical features of the tristetraprolin zinc finger domain. zfs1 has been linked to pheromone signal transduction control and to the coordination of mitosis, but no biological function has been ascribed to the zfs1 protein. Through a functional genomics approach we compared transcript levels in wild-type and zfs1-deficient S. pombe strains; those elevated in the zfs1-deficient strain were examined for the presence of potential tristetraprolin-like binding sites. One such potential target transcript was encoded by arz1, a gene encoding a protein of unknown function that contains armadillo repeats. arz1 mRNA decay was inhibited in the zfs1-deficient strain when it was expressed under the control of a thiamine-repressible promoter. Mutations within one AU-rich element present in the arz1 3’-untranslated region protected this transcript from zfs1-promoted decay, whereas mutating another potential binding site had no effect. Binding assays confirmed a direct interaction between zfs1 and arz1 mRNA-based probes; this interaction was eliminated when key residues were mutated in either zfs1 zinc finger. zfs1 and its targets in S. pombe represent a useful model system for studies of zinc finger protein/AU-rich element interactions that result in mRNA decay. Keywords: knockout comparison, steady-state analysis
Project description:Analysis of splicing defects in Schizosaccharomyces pombe upon chemical genetic inhibition of splicing kinases dsk1, lkh1, and prp4, as well as alanine-mutation of phosphorylated residues in the splicing factors bpb1, prp2, rsd1, srp1, srp2, usp101, usp103, sum3, prp22, cdc5, and cwf22. This study shows the splicing kinase dsk1 modulates splicing efficiency of introns with non-consensus splice sites, likely through phosphorylation of bpb1. Modulation of splicing efficiency of transcripts through kinase signaling pathways may afford the necessary flexibility to tune the gene expression profile in response to environmental and developmental cues.
Project description:The phosphorylation of proteins modulates various functions of proteins and plays an important role in regulation of cell signaling. In the recent years, the label-free quantitative (LFQ) phos-phoproteomics has become the powerful tool to analyze the phosphorylation of proteins within the complex samples. Despite the great progress, the studies of protein phosphorylations are still limited in throughput, robustness, and reproducibility, hampering analyses that involve multiple perturbations, such as those needed to follow the dynamics of phosphoproteomes. To address these challenges, we introduce here the LFQ phosphoproteomics workflow that is based on Fe-IMAC phosphopeptide enrichment followed by strong anion exchange (SAX) and porous graphitic carbon (PGC) fractionation strategies. We applied this workflow to analyze the whole-cell phosphoproteome of the fission yeast Schizosaccharomyces pombe. Using the strategy, we identified 8353 phosphosites from which 1274 were newly identified. This provides the sig-nificant addition to the S. pombe phosphoproteome. Results of our study highlight that combining of PGC and SAX fractionation strategies substantially increases the robustness and specificity of LFQ phosphoproteomics. Overall, the presented LFQ phosphoproteomics workflow opens the door for studies that would get better insight into the complexity of the protein kinase functions of the fission yeast S. pombe.
Project description:RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its role in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.
Project description:During the early steps of snRNP biogenesis, the Survival of Motor Neuron (SMN) complex acts together with the methylosome, an entity formed by the pICln protein, WD45 and the PRMT5 methyltransferase. To expand our understanding of pICln and SMN functional relationships in vivo, we performed a genetic analysis of an uncharacterized S. pombe pICLn homologue. Although not essential, the S. pombe ICln protein is important for optimal yeast cell growth. The human pICln gene complements the icln∆ slow growth phenotype demonstrating that the identified SpICln sequence represents the bona fide human homolog. Consistent with the role inferred for human pICln using in vitro experiments, we found that the SpICln protein is required for optimal production of the spliceosomal snRNPs and for efficient splicing in vivo. Genetic interaction approaches demonstrate furthermore that modulation of ICln activity is unable to compensate for defects induced by SMN mutations, and reciprocally. Using a genome-wide approach and RT-PCR validation tests, we show also that splicing is altered differentially in icln∆ cells. Our data are consistent with the emerging view that splice site selection and spliceosome kinetics is highly dependent on the concentration of core spliceosomal components.