Aminoacyl tRNA-synthetases as regulators of post-transcriptional gene expression in yeast

Aminoacyl tRNA-synthetases (aaRS) comprise a family of proteins known primarily for catalyzing the aminoacylation of their cognate tRNA, necessary for translation.
In addition to their canonical function of tRNA aminoacylation, many divergent, nonconventional roles for these proteins are known. Specifically, it was shown that some aaRS can bind a specific mRNA. Previous work in our lab identified the mRNA bound by five aaRS in yeast (S. cerevisiae). Further study on one of them (HisRS) revealed a resemblance between its target site and its tRNA anticodon stem-loop. It was also found that HisRS binding has a role in translation control. This project describes the transcriptome-wide mRNA association of the vast majority of S.cerevisiae cytosolic aaRSs. To achieve this, we tagged each aaRS with GFP and performed an RNA immunoprecipitation protocol for each cytosolic aaRS, followed by next-generation sequencing (RIP-Seq). The sequencing results will help us identify tRNA-mimic sites involved in aaRS binding of mRNA, as well as families of mRNA bound by aaRS which might suggest other roles related to post-translational control, such as amino acid synthesis.
Furthermore, since HisRS was found to regulate the translation of its target mRNA, the need for additional RNA-Binding proteins involved in regulation is examined. For that, we isolate target mRNA by using MS2-loop tags as a handle. These mRNAs are captured by MS2-binding protein beads. Associated proteins are identified by tandem mass spectrometry. Translation factors among these will be tested further to define their role in regulating translation upon aaRS binding.