Final Report Summary - COREMEN (Cross-talk of cell metabolism and post-transcriptional control via RNA-binding enzymes)
Ribonucleic acid (RNA)-binding proteins (RBPs) determine RNA fate from synthesis to decay. Against this background it is unsurprising that numerous diseases have been linked to defects in RBP expression and function, including neuropathies, muscular atrophies, metabolic disorders and cancer. We have developed a method, called interactome capture, which allows isolation of 'all' RBPs bound to messenger RNAs (mRNAs) in proliferative HeLa cells. The HeLa mRNA interactome is composed by 860 proteins and adds more than three hundred RBPs to those previously known. Our data have been experimentally validated and shed new light on diverse aspects of RNA biology, including RBPs in disease, RNA-binding kinases and RNA-binding architectures:
(i) Novel RNA-binding domains: We have discovered three novel globular RNA-binding domains (RAP, AKAP95 and HC5HC2H Znf) and we have extended the RNA-binding activity to several domains with sparse evidence to bind RNA (e.g. SAP and WD40). Moreover, we have found that large portions of the protein within the HeLa mRNA interactome are intrinsically disordered, natively lacking stable three-dimensional structure and enriched in repetitive motifs (e.g. RGG boxes and long poly(K) patches) that could serve as a platform for RNA-binding. The high degree of conservation in numerous non-homologous RBPs point towards an emerging role of such intrinsically disordered domains in RNA biology.
(ii) RBPs in human diseases: Eighty six proteins of the HeLa mRNA interactome are listed in the online Mendelian inheritance in man (OMIM) database as being associated with human Mendelian diseases, and most of these (48) were previously unknown to be RBPs. Disturbances of RNA metabolism can now be explored for these 48 proteins to further understand their roles in the respective human disorders.
(iii) RNA-binding metabolic enzymes: The HeLa mRNA interactome contains 17 metabolic enzymes that cover much of the landscape of intermediary metabolism and appear not to cluster into particular pathways. Furthermore, we have explored using next-generation sequencing the scope of RNAs bound by enolase 1 and serine-hydroxymethyl transferase 2, revealing hundreds of mRNAs, most of them involved in gene expression. These striking results suggest that these metabolic enzymes could control the cellular gene expression program by regulating particular set of mRNAs at the posttranscriptional level, most probably in response to intra or extracellular stimuli.
The mRNA interactome capture methodology was developed during the timeframe of this Marie Curie IE fellowship to generate the first comprehensive atlas of mRNA mRBPs of a living cell, and can now be used or adapted to study the mRNA interactomes of other cells and organisms. The approach can also be applied to investigate mRBP dynamics as a function of different biological conditions, such as metabolic changes, differences in cell growth/the cell cycle, forms of stress (hypoxia, oxidative stress, nutrient deprivation, etc.), developmental and differentiation stages, or in response to drugs. In fact, Interactome capture is already being applied by our and other laboratories (collaborators) to distinct cell types such as Saccharomyces Cerevisiae, human hepatocytes, mouse stem cell, macrophages and cardiomyocites. Therefore, Interactome capture will be broadly used and could offer unprecedented insights into biological states, complementing analyses of transcriptomes and proteomes and opening new avenues in RNA biology and molecular medicine. Moreover, this work revealed the moonlighting role of several metabolic enzymes as RBPs. If functionally relevant, as proposed by the REM (RNA, enzyme, metabolite) network hypothesis (Hentze and Preiss, 2010), these proteins could broadly connect intermediary metabolism with RNA biology and posttranscriptional gene regulation that calls for further exploration.
(i) Novel RNA-binding domains: We have discovered three novel globular RNA-binding domains (RAP, AKAP95 and HC5HC2H Znf) and we have extended the RNA-binding activity to several domains with sparse evidence to bind RNA (e.g. SAP and WD40). Moreover, we have found that large portions of the protein within the HeLa mRNA interactome are intrinsically disordered, natively lacking stable three-dimensional structure and enriched in repetitive motifs (e.g. RGG boxes and long poly(K) patches) that could serve as a platform for RNA-binding. The high degree of conservation in numerous non-homologous RBPs point towards an emerging role of such intrinsically disordered domains in RNA biology.
(ii) RBPs in human diseases: Eighty six proteins of the HeLa mRNA interactome are listed in the online Mendelian inheritance in man (OMIM) database as being associated with human Mendelian diseases, and most of these (48) were previously unknown to be RBPs. Disturbances of RNA metabolism can now be explored for these 48 proteins to further understand their roles in the respective human disorders.
(iii) RNA-binding metabolic enzymes: The HeLa mRNA interactome contains 17 metabolic enzymes that cover much of the landscape of intermediary metabolism and appear not to cluster into particular pathways. Furthermore, we have explored using next-generation sequencing the scope of RNAs bound by enolase 1 and serine-hydroxymethyl transferase 2, revealing hundreds of mRNAs, most of them involved in gene expression. These striking results suggest that these metabolic enzymes could control the cellular gene expression program by regulating particular set of mRNAs at the posttranscriptional level, most probably in response to intra or extracellular stimuli.
The mRNA interactome capture methodology was developed during the timeframe of this Marie Curie IE fellowship to generate the first comprehensive atlas of mRNA mRBPs of a living cell, and can now be used or adapted to study the mRNA interactomes of other cells and organisms. The approach can also be applied to investigate mRBP dynamics as a function of different biological conditions, such as metabolic changes, differences in cell growth/the cell cycle, forms of stress (hypoxia, oxidative stress, nutrient deprivation, etc.), developmental and differentiation stages, or in response to drugs. In fact, Interactome capture is already being applied by our and other laboratories (collaborators) to distinct cell types such as Saccharomyces Cerevisiae, human hepatocytes, mouse stem cell, macrophages and cardiomyocites. Therefore, Interactome capture will be broadly used and could offer unprecedented insights into biological states, complementing analyses of transcriptomes and proteomes and opening new avenues in RNA biology and molecular medicine. Moreover, this work revealed the moonlighting role of several metabolic enzymes as RBPs. If functionally relevant, as proposed by the REM (RNA, enzyme, metabolite) network hypothesis (Hentze and Preiss, 2010), these proteins could broadly connect intermediary metabolism with RNA biology and posttranscriptional gene regulation that calls for further exploration.