A Cytoplasmic Complex Mediates Specific mRNA Recognition and Localization in Yeast
Figure 8
She3p does not play a role in the nucleus.
(A) Immunofluorescence stainings of HA-tagged She3p in the temperature-sensitive nuclear export mutant mex67-5. At permissive temperature (25°) She3p was localized to the bud tip (upper panels). At restrictive temperature (37°) nuclear export is blocked, but She3p still remained in the cytoplasm (lower panels; position of nuclei marked by arrowheads). It indicates that She3p does not shuttle into the nucleus. At restrictive temperature, She2p is trapped in the nucleus and mRNA as well as Myo4p localization is abolished [16], which is consistent with the diffuse cytoplasmic distribution of She3p. Bar: 5 µm. (B) RNase sensitive She2p occupancy at different genes in wild-type and mutant yeast cells lacking She3p. She2p is recruited co-transcriptionally to genes coding for localized and non-localized transcripts, as it was reported recently [20]. She2p occupancy increases towards the 3′-end of genes. There are no significant differences of She2p occupancy in wild-type and Δshe3 cells (two-tailed t test p values>0.05 for all genomic regions tested). An RNase treatment prior to the immunoprecipitation step led to a clear reduction of She2p occupancy, indicating an association with nascent mRNA. The most severe effect was observed at the 3′-end of genes (ASH1, ADH1: 30%–40% of reduction). Again, no significant differences could be observed between wild-type and mutant yeast cells. RNase treatment affected She2p occupancy at genes coding for localized and non-localized transcripts in a similar way. ChIP occupancies are shown for three different regions of ASH1 (all primer pairs are located within the ORF region) and ADH1, as well as for a single region of PMA1 and FBA1 as indicated on the x-axis. The fold enrichments over an ORF-free heterochromatic region on chromosome V are shown on the y-axis. Error bars express the standard deviation from two independent experiments of biological replicates. TSS, transcription start site; ORF, open reading frame; pA, polyadenylation site.