The interconnections between intermediary metabolism and regulation of gene expression are relatively poorly understood. A few reports have analysed the ability of some metabolic enzymes to 'moonlight' as RNA-binding proteins, being responsive to small metabolites that often represent co-factors of the enzymes. Building on these observations, it was recently proposed that regulatory networks formed between mRNAs, Enzymes and Metabolites (REM networks) might represent a novel regulatory layer interconnecting intermediary metabolism and post-transcriptional regulation of gene expression.
This project aimes to comprehensively address the largely unexplored territory of cross-talk between intermediary metabolism and post-transcriptional gene regulation by identifying mRNA-binding proteins in living yeast cells using unbiased proteomic and transcriptomic methods. By adaptation of a protocol established in the host lab for human cells, I will employ in vivo crosslinking and oligo d(T)-purification followed by mass spectrometry to create a catalogue of mRNA-binding proteins for yeast, thereby identifying enzymes conserved between human and yeast. To find 'target' RNAs that specifically interact with these enzymes, I will use crosslinking and immunoprecipitation (CLIP/CRAC) assays followed by high-throughput RNA sequencing. This will allow me to identify the interacting motifs in both, the RNA and the enzymes by bioinformatic and biochemical methods. Finally, I will explore the importance of the mRNA-protein interactions for gene regulation using functional assays in vivo and in vitro.
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