Posttranscriptional regulation of cell-cell interaction protein-encoding transcripts by Zfs1p in Schizosaccharomyces pombe

Abstract

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 zfs1Δ 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 zfs1Δ mutants; a subset of these transcripts decayed more slowly in the zfs1Δ 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 zfs1Δ cbf12Δ double mutants demonstrated that the increased flocculation seen in zfs1Δ 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.

Fig 1

Polyadenylation site determinations for the WT and zfs1Δ mutants. Direct RNA sequencing was performed using total RNA purified from two independent isolates of WT and zfs1Δ mutants. Data shown are from 7 transcripts elevated for the zfs1Δ mutant (A to G), a transcript downregulated for the zfs1Δ mutant (H), and the zfs1 transcript itself (I). The horizontal axis represents base pairs 3′ of the stop codon. The arrows indicate the locations of 7-mer/9-mer potential Zfs1p binding sites.

Fig 2

Flocculation analysis of zfs1Δ and cbf12Δ mutants. (A) Flocculation of WT and zfs1Δ cells (A) and flocculation of WT, zfs1Δ, cbf12Δ, and zfs1Δ cbf12Δ cells (B) was initiated by the addition of CaCl₂ (+ Ca) and determined by using the Helm assay (35). The percentage of cells in suspension was measured based on the optical density. Shown are the means ± standard errors of the means of values from three independent experiments.

Fig 3

Real-time RT-PCR analysis of target transcripts. Poly(A)⁺-specific mRNA was extracted from WT, zfs1Δ, cbf12Δ, and zfs1Δ cbf12Δ cells and subjected to real-time RT-PCR with gene-specific primers. Shown are the mean values ± the standard errors of the means (from three independent experiments) of relative mRNA abundance normalized to an internal control, gapdh.

Fig 4

Decay analysis of Zfs1p target transcripts. WT and zfs1Δ strains were transformed with plasmids containing the indicated genes under the control of the nmt promoter, followed by 500 bp of the mRNA 3′-UTR. The strains were grown in medium lacking thiamine, to allow the expression of the plasmid-derived transcripts Spbc359.04c (A), Spcc1494.03 (B), Spbc8E4.12c (C), Spac6B12.02c (D), Spac4F8.10c (E), Spac589.03c (F), Spcc1223.13 (G), and Spbc3E7.02c (H). A 10 μM concentration of thiamine was then added to the medium, and samples were taken at the indicated times for real-time RT-PCR analysis. Shown are the mean values (± the standard errors of the means) of the relative mRNA abundance normalized to an internal control, gapdh, from three independent experiments.

Fig 5

RIP analysis of Zfs1p binding to target transcripts. RNA immunoprecipitation was performed with strains expressing a C-terminal HA-tagged Zfs1p and a no-tag control. Proteins and associated RNA were precipitated with anti-HA antibodies, analyzed using real-time RT-PCR, and normalized to the input amount of RNA. Shown are the mean values (± the standard errors of the means) of the relative abundance of immunoprecipitated (IP) RNA normalized to the input (IN) RNA from three independent experiments.