The Study On The Cross-Talk Of Phosphorylation And PARylation Of ARE Binding Protein ELAV1/HuR

Controlled mRNA turnover is of great importance for many cellular processes,particularly in post transcriptional regulation of gene expression and contributed to mammalian cells responding to different damaging stimuli.This processe is efficiently influenced by a diverse group of RNA-binding proteins(RBPs),which associate with subsets of mRNAs that share specific sequences.Among numbers RBPs,the ARE binding proteins are the most studied group of trans-acting mRNA stability determinants.Many labile mRNAs have relatively long 3’-untranslated regions(UTRs)featuring characteristic AU-rich stretches that are collectively known as AREs.Control of selective mRNA degradation mediated by AU-rich elements(AREs)encoding oncoproteins,cytokines and transcription factors is a paradigm for post-transcriptional regulation[3].The stability of AU-rich mRNAs is thought to be controled by those AREs binding proteins(AUBPs).Most of AUBPs act to degrade mRNA,such as AUF1,hn RNP D,TTP,BRF1 and KSRP,whereas fewers selectively stabilize mRNA,the best known that stabilize and in some instances influence the translation of ARE-bearing mRNAs are the members of the Hu family.There are four proteins belonging to the Hu family.HuR(Hu A)is expressed ubiquitously in all tissues,whereas Hu B(or Hel-N1),Hu C,and Hu D are neuron-specific.HuR protein has numerous functions mostly related to cellular stress response,such as cell differentiation,carcinogenesis,and responses to stress and immune stimuli.All Hu proteins contain three RNA binding domains(RRMs),RRM1 and RRM2 positioned next to each other and followed by a hinge region and a terminal RRM3,among all four Hu family proteins the RRMs are conserved,whereas the hinge region between RRMs 2 and 3 differs.The HuR’s HNS(HuR nucleocytoplasmic shuttling sequence),which contains both a nuclear localization signal and a nuclear export signal.The modification of different sites of HNS domain(especially phosphoserine)has been found to influence HuR’s shuttling between cytoplasm and nuclear,since HuR is localized predominantly in the nucleus.The HNS of HuR has been reported to be phosphorylated by many kinases,such as DNA damage response kinases,Cdk1,Chk2,PKC at several serine positions,this phosphoserine modification was regarded as the most important factor to cause HuR shuttling and run its function in cytoplasm.Just like methylation or phosphorylation,poly ADP-ribosylation(PARylation),is one of another essential post-translational protein modifications in cells.By which,PARP family using NAD+ as a substrate synthesizes poly ADP-ribose units(PAR)in linear or branched and bind the polymers of ADP-ribose to acceptor proteins(PARylation).PARP 1 as the most abundant and ubiquitous member of the family,accounts the most of cellular PARP activity.Although PARP1 is the major target of its own activity,other covalently PARylated proteins,including histones and transcription factors have been described.PARylation influences the activity of target proteins by modulating their protein-nucleic acid interactions,enzymatic activity,protein-protein interactions,or subcellular localization through an modification reaction.Our previous studies have demonstrated that PARP1 through PARylation of RNA binding protein HuR mediated mRNA stability after LPS exposure.Moreover,the D226 in HNS domain is the important site to be PARylated and this kind of PARylation may influence HuR phosphoserine.Here,we systematically examined if modification of PARylation could influence the phosphorylation of serines sites within the hinge region of HuR.Our results identify the Sxxx of HNS phosphoserine,as being important for HuR localization,is controlled by D226 site PARylation.All this imply that PARylation could influence phosphoserine of HuR’s HNS domain regulate HuR location and functions.This study aims to clarify the interaction between poly(ADP-ribose)of HuR and HuR phosphorylation,find the phosphorylation sites involved and the signaling pathways,as to add new content to the regulation of HuR post translation.At the same time,it provides a new therapeutic target for the treatment of diseases with high level of post transcriptional regulation in the pathological process.