RNA-binding Protein HnRNPR Reduces Cholesterol Levels By Binding To And Suppressing HMGCR

Heterogeneous nuclear ribonucleoprotein(hnRNPs)represent a large family of RNA-binding proteins that are involve in myriad of functions such as nuclei acid metabolism,alternative splicing,mRNA stabilization,transcriptional and translational regulation.Recent studies have demonstrated and identified diverse array of RBPs that are associated with lipid and cholesterol metabolism and its associated cardiovascular disorders.However,the role of hnRNPR in cholesterol metabolism and homeostasis in neurons,whether it has a role in the regulation of HMGCR is largely unknown.This study for the first time identifies hnRNPR as a repressor of HMGCR,a well-studied rate limiting,and irreversible enzyme in the de novo synthesis of cholesterol.In our current work,we first performed amplex red cholesterol assay to ascertain if hnRNPR has the potential to modulate relative cholesterol level.Our assay demonstrated that hnRNPR can modulate cholesterol by up and downregulation.We next,sought to determine the binding affinity and binding motifs of hnRNPR with cholesterol biosynthetic genes.Therefore we screened all enzymes that are involved in cholesterol biosynthesis pathway that have binding motif of hnRNPR by checking the mRNA and proteins levels of these genes in hnRNPR overexpression and knockdown samples.The rationale for doing this is to enable us ascertain the actual genes that have binding motifs of hnRNPR as this will help us to leverage on the next set of genes or experiments to focus on.Using a genome browser,the following cholesterol biosynthetic genes were identified:CYP51A1,FDFT1,HMGCR and NSDHL,and were subsequently screened by checking their expression levels of mRNA and protein levels respectively.Out of the screened genes,only HMGCR show a significant upregulation and down-regulation of mRNA and protein levels when hnRNPR is knockdown or overexpressed respectively and is subsequently considered for further study.Knockdown and overexpression of hnRNPR in cultured neuroblastoma cell line(Neuro-2a)and MN1 cell lines enhance and inhibit HMGCR in vitro,respectively.hnRNPR may exert its repressive activity on HMGCR mRNA and protein levels by using its RRM in recognizing and modulating the stability of arrays of HMGCR transcripts.Our RNA immunoprecipitation assays demonstrate a interaction between hnRNPR and HMGCR mRNA.We found that hnRNR binds to the 3'untranslated region(3'UTR)of HMGCR and reduces its translation,while hnRNPR silencing increases HMGCR expression and cholesterol metabolism in N2a and mnl cells.Rescue assay was also performed for repressed cholesterol levels in hnRNPR overexpression via amplex cholesterol enzyme assay,which was rescued by HMGCR construct supplementation.Taken together,we have identified hnRNPR as a novel post-transcriptional regulator of HMGCR expression in neuronal cholesterol homeostasis.These data suggest that modulation of cholesterol by hnRNPR could open a new vistas in understanding the physiology,pathogenesis and pharmacotherapy of cholesterol related disorders caused by cholesterol imbalance.