Mechanisms regulating mRNA localization and localized translation

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The cytoplasmic transport and localization of mRNA is a post-transcriptional mechanism that restricts the synthesis of proteins to specific sites within a cell. This process plays important roles in the development of multicellular organisms, in cell motility, in asymmetric cell division and in neuronal growth. We are using the budding yeast Saccharomyces cerevisiae, in which over thirty mRNAs are localized to the bud tip, as a model organism, as it offers the advantage of a simple and genetically tractable organism, which possesses the same underlying mechanisms involved in mRNA localization and translational control as in metazoans. In the current model of mRNA localization in budding yeast, bud-localized transcripts are first recognized in the nucleus by the RNA-binding protein (RBP) She2, which binds their localization element(s). Translational repressors are also recruited on the mRNA in the nucleus. Once in the cytoplasm, She2 binds the adaptor protein She3 and the type V myosin Myo4, forming a complex transported along the actin cytoskeleton toward the bud tip. Once localized, phosphorylation releases the translational repression on the mRNA, allowing its local translation. Recently, a phosphoproteomic analysis of She2 revealed the presence of several phosphorylated residues, including CK2-dependent phosphorylation sites in the NLS of She2. Some of these phosphorylation events inhibit the dimerization of She2, supporting a potential regulatory role for these post-translational modifications. We also found that She2 is recruited cotranscriptionally to bud-localized transcripts via its interaction with the RNA polymerase II elongation factor Spt4-Spt5. It remains unclear how She2 interacts with Spt4-Spt5 and why this interaction is required. We hypothesize that the interaction between She2 and Spt4-Spt5 modulates the affinity of She2 for binding nascent RNA zipcodes. Phosphorylation of She2 would regulate its nuclear import and access to nascent RNA zipcodes. To explore these questions, we propose the following objectives: 1) Determine how She2 is recruited to its target mRNAs during transcription. We will explore the roles of Spt4-Spt5 and Loc1 in the recruitment of She2 on nascent transcripts. ChIP-seq experiments will help determine the extent of She2 and Loc1 cotranscriptional recruitment genome-wide. 2) Identify the roles of She2 phosphorylation in the regulation of mRNA localization. We will explore the role of CK2-dependent phosphorylation of She2 in regulating its nuclear import and activity. We will use proteomics to identify and characterize other kinases acting on She2. This program will reveal important mechanistic insights on the role of the transcription machinery in the cotranscriptional recruitment of RBPs involved in mRNA localization. Furthermore, new levels of regulation of these processes will be explored, which will help understand how cells control, in space and time, the local synthesis of their proteins.