Posttranscriptional Regulation of Cell-Cell Interaction Protein-Encoding Transcripts by Zfs1p in Schizosaccharomyces pombe

Abstract

f 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. In this study, we identified probable Zfs1p target mRNAs by comparing transcript levels in wild-type yeast and zfs1⌬ mutants, using deep sequencing and microarray approaches. We also used direct RNA sequencing to determine polyadenylation site locations and to confirm the presence of potential Zfs1p target sequences within the target mRNA. These studies identified a set of transcripts containing potential Zfs1p binding sites that accumulated significantly in the zfs1⌬ mutants; a subset of these transcripts decayed more slowly in the zfs1⌬ mutants and bound directly to Zfs1p in coimmunoprecipitation assays. One apparent direct target encodes the transcription factor Cbf12p, which is known to increase cell-cell adhesion and flocculation when overexpressed. Studies of zfs1⌬ 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. These data suggest that Zfs1p can both directly and indirectly regulate the levels of transcripts involved in cell-cell adhesion in this species.

Functional Equivalence of an Evolutionarily Conserved RNA Binding Module

“…A synthetic peptide comprising the TZF domain of human TTP bound to this 9-mer sequence with a K d of 3.2 nM at 24°C, as shown by fluorescence anisotropy (11). Remarkably similar affinities have been demonstrated for the analogous TZF domains from TTP family members from a variety of other species, including the single family members expressed in Drosophila melanogaster (5), the fission yeast Schizosaccharomyces pombe (4,7), and the human pathogenic yeast Candida albicans (6). In the experimental situations tested to date, mutation of any one of the cysteines or histidines that comprise the two zinc coordination centers of the TZF domain will abrogate RNA binding.…”

“…The deletion of zfs1 has been described previously (4). The wild-type control strain (referred to as Zfs1:FLAG, Sp TZF L1, and Sp Zfs1) was generated using the pFa6a-3ϫFLAG-6ϫGly-KanMX vector (18).…”

“…Probe sets included 46 transcripts that were chosen on the basis of our previous mRNA sequencing analysis (4). The raw counts were adjusted for technical variability using positive and negative controls included in the code sets and then normalized to internal controls identified previously.…”

“…To do this, we analyzed RNA binding affinity using purified components and fluorescence anisotropy and performed in vivo functional analyses in S. pombe. In this species, loss of the single TTP family member Zfs1 results in the abnormal accumulation of several dozen transcripts, many of which have been shown to be bound directly to Zfs1 in coimmunoprecipitation analyses and which decayed more slowly in zfs1 deletion cells (4,7). Many of these target transcripts encode proteins involved in cell-cell adhesion, and deletion of zfs1 caused a severe flocculation defect.…”

The Drosophila Tis11 Protein and Its Effects on mRNA Expression in Flies

“…At least one of the two such proteins expressed in Saccharomyces cerevisiae affects the stability of a collection of transcripts involved in regulating iron metabolism, apparently also by binding to ARE elements that are similar to their mammalian counterparts (20,21). In Schizosaccharomyces pombe, the single family member, Zfs1p, also binds to and promotes the decay of ARE-containing mRNAs (22,23), but in this case, the major phenotypic effect of the protein is to prevent abnormal cell-cell interactions, and iron metabolism appears to be unaffected. This evidence strongly suggests that at least some of the functions of proteins from this family have been conserved in eukaryotes, such as the ability to bind to and destabilize ARE-containing mRNAs, but that these proteins can be used for different physiological purposes in different species.…”