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REM networks in yeast

Final Report Summary - YREM (REM networks in yeast)

REM networks in yeast (yREM)

Cells must respond to external (e.g. hypoxia, nutrient levels) and internal (e.g. cell cycle) stimuli and adapt their gene expression profiles as well as their intermediary metabolic pathways accordingly. Therefore, regulation of eukaryotic gene expression is spatially and temporally tightly controlled. During the last two decades, a variety of metabolic enzymes have been found to possess RNA-binding activity. Examples of such enzymes which 'moonlight' as mRNA-binders have been described in different biochemical pathways such as glyceraldehyde-3- phosphate dehydrogenase (GAPDH; glycolysis), lactate dehydrogenase, phosphoglycerate kinase (pentose cycle), inosine monophosphate dehydrogenase (nucleotide synthesis), enoyl-CoA- hydratase (fatty acid metabolism), aconitase 1 (tricarboxilic [TCA] cycle), catalase and several more. However, the biological role of this moonlighting activity of metabolic enzymes remains poorly understood and for most of these enzymes no physiological role in cellular regulation of mRNA expression has been defined so far.

The possibility that regulatory networks consisting of RNA, Enzymes and their Metabolites (or 'REM-networks') may serve to coordinate gene regulation and metabolism in vivo has recently been proposed by Matthias Hentze and Thomas Preiss. We adapted the mRNA interactome capture protocol for identifying mRNA-binding proteins in mammalian cells (established in the laboratory of Matthias Hentze, EMBL Heidelberg, Germany) to identify mRNA-binding proteins (RBPs) of the unicellular yeast Saccharomyces cerevisiae. The technique includes in vivo UV crosslinking to freeze RNA-protein interactions, stringent purification and poly-A RNA selection via oligo d(T) magnetic beads and mass spectrometry of the purified proteins.
Using quantitative proteomics, we identified 678 high confidence (m)RBPs (FDR<0.01) including many previously unknown ones. Our data set includes 101 of 120 recently reported yeast RBPs, and additionally identifies 283 high confidence RBPs.
Moreover, integration of our data with published work on mammalian RBPs defines a first eukaryotic „core interactome“ of more than 250 RBPs that are conserved between mammals and yeast. We analysed the evolutionary conserved attributes of these ancient RNA-binders, as well as 'mammalian-only' and 'yeast-only' features of RBPs.

Strikingly, the core interactome includes several RBPs involved in biochemical pathways, particularly of central carbon metabolism, largely expanding our knowledge about moonlighting enzymes beyond individual single reports.
RNA-binding of these enzymes is poorly characterized to date; we utilized two complementary novel approaches to identify the RNA-binding domains of these exceptional RBPs providing us with a unique dataset of RNA-protein interaction sites. We are now using this information to mutate enzymes to distinguish enzymatic activity from RNA-binding. The advanced genetic tools in yeast to completely replace a genomic coding sequence by a mutated- or tagged version of the enzyme will help us to understand the role of RNA-binding in enzymes of intermediary metabolism and guide our advancement to understand REM networks in eukaryotes.

The research project 'REM networks in yeast' (yREM) was supported by a Marie Curie Fellowship (FP7/2007-2013 / MC-IEF-301031).