High-throughput Mass Spectrometric Identification Of The RNA-binding Proteins In Subcellular Regions

Background and objective: Interactions between RNA and RNA-binding proteins(RBPs)play important roles in the regulation of gene expression as well as many other biological processes.Dysfunction of RBPs has been linked to a number of human diseases.The finding of unknown RBPs is essential to investigate the RNA associated biological processes and molecular mechanisms.However,there are certain limitations in several published methods of RBPs identification,which makes it difficult to satisfy the requirements of sensitivity and accuracy in the identification of RBPs on a high-throughput level.Thus,we developed a method to identify RBPs in different subcellular regions and to detected the RNA-binding regions by combining UV crosslinking,acidic guanidinium-thiocyanate-phenol-chloroform(AGPC)biphasic extraction and quantitative mass spectrometry.Methods: We developed SFABE(subcellular fractionation and AGPC biphasic extraction),a method combining UV crosslinking,subcellular fractionation and AGPC biphasic extraction to purify the RBPs from nucleus and cytoplasm,and then analyze them with liquid chromatography-tandem mass spectrometry(LC-MS/MS).We next compared the SFABE RBPs with the data of other high-throughput methods,and performed a series of analysis of protein features,including isoelectric point(PI),molecular mass and hydrophobicity.We then performed the GO and KEGG pathway analysis of the RBPs identified from different subcellular fractions.To validate the performance of SFABE,we examined six putative RBPs by UV-crosslinking assisted western blotting.In addition,we purified and detected the adjacent peptides of crosslinked sites,and computationally extrapolates the RNA-binding regions.Results: By using SFABE,1783 and 2251 RBPs were identified from MDA-MB-231 cell nucleus and cytoplasm,respectively.SFABE RBPs covered more than two-thirds of the GO-annotated RBPs and significantly overlapped with the data of other high-throughput methods.Bioinformatic analysis revealed that SFABE RBPs were specifically enriched in RNA-related function pathways.Moreover,we found that 202 RBPs,which act as metabolic enzymes,participated in a variety of metabolism pathways,including carbohydrate,amino acid and lipid metabolism.UV-crosslinking assisted western blotting analysis showed that all the six metabolic enzymes could bind to RNAs.We identified 2873 RNA-binding regions within 1090 RBPs.710,1160 and 1003 binding sites were mapped to known RBDs,unknown RBDs and intrinsically disordered regions(IDRs),respectively.Conclusions: We developed a high throughput method termed as SFABE to purify and identify RBPs in different subcellular regions.By using this method,we confirmed many earlier reported RBPs and identified hundreds of novel ones.The cellular localizations of the identified RBPs were documented,showing distinct biological functions RBPs in different subcellular regions.Our findings markedly increased the number of RBPs and demonstrated the RNA-binding capability of numerous metabolic enzymes.Furthermore,we characterized 2873 RNA binding interfaces,providing crucial structural information regarding the complex nature of RNA-protein interactions.