Hepatocytic mRNA interactome

mRNAs are regulated at multiple steps throughout their lifetime by RNA-binding proteins (RBPs) which collectively represent the “mRNA interactome” of a cell. Interestingly, many unexpected proteins such as metabolic enzymes were found to bind mRNAs in vivo [1, 2], potentially playing important roles interconnecting posttranscriptional regulation with cellular metabolism [3]. IRP1/aconitase represents a well studied example, which, depending on intracellular iron levels, switches between its functions as an enzyme and an RBP controlling mRNAs involved in iron metabolism. As liver cells represent a metabolically critical cell type, we here report the determination of the mRNA interactome of HuH-7 hepatocellular carcinoma cells. We employed two complementary in vivo RNA-protein crosslinking protocols, isolated the mRNP complexes by oligo-d(T) purification and analyzed purified proteins by quantitative mass spectrometry (MS). We identified 726 proteins, including known RBPs, expected RBPs which are expressed in the hepatic cells, and many previously unknown RBPs. Interestingly, especially enzymes of central energy metabolism pathways display mRNA binding, notably of the glycolytic pathway and the TCA cycle. To uncover dynamic responses of the mRNA interactome to altered metabolic conditions, cells were treated with glycolytic inhibitor 2-deoxyglucose or left untreated prior to interactome capture and comparative quantitative MS. We will report the development of interactome capture as a novel experimental approach to characterize the biological states of cellular systems and the responses of the mRNA interactome to altered glycolysis.

Literature:

1. Castello, A., et al.: Insights into RNA Biology from an Atlas of Mammalian mRNA-Binding Proteins. Cell, 2012

2. Baltz, A.G., et al.: The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol Cell, 2012

3. Hentze, M.W. and T. Preiss: The REM phase of gene regulation. Trends Biochem Sci, 2010