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
