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Characterization of zfs1 as an mRNA binding and destabilizing protein in Schizosaccharomyces pombe

ABSTRACT: 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

ORGANISM(S): Schizosaccharomyces pombe Saccharomyces cerevisiae

PROVIDER: GSE8122 | GEO | 2007/11/28

SECONDARY ACCESSION(S): PRJNA100999

REPOSITORIES: GEO

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Project description:Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins can bind directly to AU-rich elements in mRNAs and promote transcript deadenylation and decay. The yeast Schizosaccharomyces pombe expresses a single TTP family member, Zfs1p, that has been linked to the mating response pathway and septum formation. We showed previously that Zfs1p can bind to and promote the destabilization of AU-rich element-containing transcripts. In this study, we identified additional target transcripts by comparing transcript levels in wild type and zfs1 mutant yeast, using deep sequencing and microarray approaches. We also used direct RNA sequencing to determine the locations of the polyA tails in both wild type and mutant strains, and to confirm the presence of potential Zfs1p target sequences within the mRNA. These studies identified a set of transcripts containing potential Zfs1p binding sites that accumulated significantly in the zfs1 mutants; a subset of these turned over more slowly in the zfs1 mutant strain, and bound directly to Zfs1p in co-immunoprecipitations. One apparent direct target encodes the transcription factor Cbf12p, which is known to increase cell-cell adhesion and flocculation when over-expressed. Studies of zfs1 and cbf12 double mutants demonstrated that the increased flocculation seen in zfs1 mutants is due, at least in part, to a direct effect on the turnover of cbf12 mRNA, leading in turn to changes in the levels of its transcriptionally regulated genes. These data suggest that Zfs1p can both directly and indirectly regulate the levels of transcripts involved in cell-cell adhesion in this species. Indicated strains were grown in YES media to approximately OD600=1.0 and total RNA was harvested, followed by polyA+ purification. Three independent microarrays were performed: 1) Project 488 included 3 biological replicates each of WT and zfs1M-bM-^HM-^F and compared PolyA+ RNA, 2) Project 529 included 5 biological replicates each of WT and zfs1M-bM-^HM-^F, and 3) Project 582 included 3 biological replicates each of WT, zfs1M-bM-^HM-^F, cbf12M-bM-^HM-^F, and zfs1M-bM-^HM-^Fcbf12M-bM-^HM-^F.

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Characterization of zfs1 as an mRNA binding and destabilizing protein in Schizosaccharomyces pombe

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