PARP1 Increases The Stability Of The Pro-inflammatory Gene MRNA Via RNA Binding Protein HuR

Poly(ADP-ribosyl)ation(PARylation)is mainly catalyzed by poly-ADP-ribose polymerase 1(PARP1),whose role in gene transcription modulation has been well established.PARP1 activation has been implicated in a wide range of biological processes,such as maintenance of genome integrity,cell death,DNA repair,and inflammatory response.Recently,studies have addressed the involvement of PARP1 activation in post-transcription regulation.For example,PARP1 has been report that regulates IP-10 expression in murine embryonic fibroblast(MEF)at a post-transcriptional level,but the mechanism has not been elucidated.Further more,poly(A)polymerase is PARylated during heat shock,leading to the inhibition of mRNA polyadenylation of target genes in a PARP1-dependent manner.These have prompted the PARP1 can participate in regulating gene expression in the post-transcription level.The post-transcription level,especially the regulation of mRNA stability,mainly regulate by cis-acting elements and the corresponding trans-acting factor.Inflammatory factor,cell factor and some proto-oncogenes mRNA are short-lived mRNA,both in its 3 ’UTR contains a AU enrichment.The AREs that commonly exist in the 3′UTRs are major mRNA destabilization determinants.With their associated binding proteins,AREs have significant physiological functions in the modulation of mRNA decay.Among ARE binding protein,Hu protein,is one of the few that have been demonstrated to stabilize ARE-containing mRNAs.The functional and shuttling regulation of HuR relies on diverse post-translational.Our previous studies reported that PARP1 binds to and modifies RelA/p65,therefore,promoting the NF-κB-dependent expression of pro-inflammatory cytokines.The expression of inflammatory genes is tightly regulated by both transcriptionaland post-transcriptional mechanisms because modifying mRNA stability provides rapid and flexible control.This urged us to question whether PARP1 regulates the stability of the mRNA transcribed from inflammatory cytokine/chemokine genes.To test whether PARP1 plays an important role in regulating gene expression at the post-transcriptional level,macrophages were exposed to lipopolysaccharides(LPS)with or without PARP1 inhibition.1)Inflammatory Cytokines & Receptors PCR arrays experiment showed LPS-induced increase in the stability of mRNAsfrom pro-inflammatory genes including chemokine(C-X-C motif)ligand 2(Cxcl2),was diminished by PARP1 inhibition/depletion,specifying the role of PARP1 in maintaining mRNA stability;2)Using Dual-reporter assays together with siRNA and PARP1 overexpressin,we demonstrated the PARP1 modulates Cxcl2 mRNA stability by acting on HuR;3)Moreover,Coimmunoprecipitation and In vitro PARylation assay showed LPS induces association of PARP1 with,and PARylation of HuR,and PARP1 PARylates HuR primarily at aspartic acid 226 upon LPS stimulation;4)Immunofluorescence assay supported the role of PARP1 in promoting HuR’s nuclear export;5)RNA-EMSA and RNA-IP assays suggested that protein PARylation increases the association of HuR with the mRNA targets;6)Further more,mRNA half life assay indicated that D226 PARylation is crucial for HuR’s function.The results presented a novel mechanism toregulate gene expression at the post-transcriptional level by PARP1 activation.