A cell-wide investigation of the RNA-binding proteins modulated by starvation and stimulation with insulin

Abstract

The development of cutting-edge proteomic techniques has recently enabled to comprehensively catalogue more than a thousand of RBPs (RNA-binding proteins) across different cell types, tissues and whole organisms. Interestingly, these methods can be employed to interrogate the effect of either cell stress or stimulation on protein-RNA interactions and can be used to uncover novel RBP candidates or new functions of well-known RBPs.This study aimed at investigating RBPs dynamics in untransformed epithelial MCF10A cells subjected to growth factor deprivation and stimulation with insulin. Both treatments have been shown to modulate RNA metabolism and primarily mRNA translation through regulation of the mTOR signalling pathway.By performing OOPS (Orthogonal organic phase separation), it was possible to identify a large number of RBPs, the interaction of which was significantly affected by starvation and stimulation with insulin, including the tumour suppressor PDCD4 (Programmed cell death protein 4).Downregulation of PDCD4 has been observed to enhance tumour progression and invasion of many different cancers. The protein is primarily a translation repressor; it impedes the physical interaction between eIF4A and eIF4G and it compromises the expression of mRNAs with highly structured 5’UTR. However, PDCD4 was also found to directly bind specific region within the coding sequence of few mRNAs and slow down their translation elongation.Although the protein harbours an N-terminal disordered region with RNA-binding potential, its relevance and its direct RNA targets have not been fully investigated in a genome-wide study. With this in mind, the PDCD4-bound transcriptome was investigated by iCLIP (Individual-nucleotide resolution UV crosslinking and immunoprecipitation) in untransformed cells and it revealed more than 150 RNA partners, the regulation of which might play a significant role during cancer development.</div