Widespread cytoplasmic mRNA transport in yeast: identification of 22 bud-localized transcripts using DNA microarray analysis

Abstract

Cytoplasmic mRNA localization provides a means of generating cell asymmetry and segregating protein activity. Previous studies have identified two mRNAs that localize to the bud tips of the yeast Saccharomyces cerevisiae. To identify additional localized mRNAs, we immunoprecipitated the RNA transport components She2p, She3p, and Myo4p and performed DNA microarray analysis of their associated RNAs. A secondary screen, using a GFP-tagged RNA reporter assay, identified 22 mRNAs that are localized to bud tips. These messages encode a wide variety of proteins, including several involved in stress responses and cell wall maintenance. Many of these proteins are asymmetrically localized to buds. However, asymmetric localization also occurs in the absence of RNA transport, suggesting the existence of redundant protein localization mechanisms. In contrast to findings in metazoans, the untranslated regions are dispensable for mRNA localization in yeast. This study reveals an unanticipated widespread use of RNA transport in budding yeast.

Fig. 1.

Schematic representation of microarray-based screens for localized RNAs. (A) Immunoprecipitations were performed with an anti-myc antibody from cellular extracts harboring either myc-tagged or untagged She proteins. RNAs enriched in pellets were amplified by RT-PCR and PCR, then labeled with either Cy5 (tagged) or Cy3 (untagged) nucleotides. Microarrays were probed with labeled PCR products, and enrichment for Cy5 was assessed. (B) Each She protein was protein A- or tandem affinity purification-tagged and affinity purified. Associated RNAs were labeled directly by reverse transcription with Cy5, then competitively hybridized on microarrays with total RNA from wild-type cells that had been labeled with Cy3. (C) Candidates from microarray analyses were tested for localization in vivo with a GFP–RNA tagging strategy.

Fig. 2.

Representative examples of localized and unlocalized RNAs identified through microarray analyses. Wild-type (SHE2, Upper) or she2Δ (Lower) cells expressing GFP–RNA for indicated transcript were visualized by fluorescence microscopy. Data for all localized RNAs are shown in Fig. 5. (Bar = 2 μm.)

Fig. 3.

Asymmetric protein localization is independent of She-based RNA transport. Wild-type (SHE2, Upper) or she2Δ (Lower) cells expressing GFP-tagged versions of the indicated proteins were visualized by fluorescence microscopy. Full results are listed in Table 1. (Bar = 2 μm.)

Fig. 4.

Coding sequences of She-transport substrates are largely sufficient for RNA localization. Wild-type cells expressing GFP–RNA for coding regions of indicated transcripts were visualized by fluorescence microscopy. Three representative transcripts are shown. Additional localized coding sequences are shown in Fig. 7, and a summary of the data is presented in Table 1. (Bar = 2 μm.)