PARP1 Participates In The Regulation Mechanism Of MRNA Stability By Promoting Oligomerization Of HuR Protein

Poly(ADP-ribosyl)ation(PARylation)is an important post-translational modification of proteins,which is mainly catalyzed by polyADP-ribose polymerase 1(PARP1).PARP1 plays an important role in various biological events through PARylation of targeted proteins.In recent years,the role of PARP1 in regulating gene expression at the post-transcriptional level has drawn increasing attention.Many studies have reported the important role of PARP1 on RNA metabolism at the post-transcriptional level,especially the effect of PARP1 on mRNA stability.The stability of mRNA is mainly regulated by specific cis-acting elements and trans-acting factors.The AU-rich sequence(ARE element)in the 3?-untranslated region(3?-UTR)of m RNA is one of the cis components that control the stability of mRNA.The main reason that ARE elements affect mRNA stability is that ARE regions can be recognized and bound by some ARE-binding proteins(ARE-BPs).Most ARE-BPs act as negative regulators,but the embryonic lethal abnormal visual-like 1(ELAVL1)/human antigen R(HuR)protein is one of the few ARE-binding proteins,which acts as a positive regulator to regulate gene expression by improving mRNA stability.The function of HuR is not only influenced by its own post-translational modification,but also by its own interaction.The latest research reported that oligomerization of HuR protein was found in clinical brain tumors and glioma cell lines.Considering that HuR protein can enhance the stability of ARE-containing mRNA,it is speculated that the oligomerization of HuR protein should be related to the stability of ARE-containing mRNA.The PARylation changes the conformation of HuR protein,which may therefore expose the sequences involved in the formation of oligomerization of HuR protein,thereby promoting the occurrence of HuR oligomerization.In this study,we further explored the mechanism of PARP1 in regulating the stability of mRNA by promoting the oligomerization of HuR under the condition of inflammatory stimulation.In our experimental system,we used tumor necrosis factor alpha(TNF?)to stimulate embryonic kidney cells 293(HEK293).In the presence of the PARP1 inhibitor Olaparib,we explored how PARP1 regulates mRNA stability through its effect on HuR oligomerization.1)We demonstrated that inflammatory stimulation can promote the occurrence of HuR oligomerization in HEK293 cells through in situchemical crosslinking analysis,immunoprecipitation and proximity ligation assay.2)In the inflammation model,we proved that the PARylation of HuR protein D226 can promote HuR oligomerization by using siRNA and D226 A site mutants.3)Next,we demonstrated that HuR oligomerization occurred in both cytoplasm and nucleus through nuclear and cytoplasmic separation experiments,and PARP1 can promote HuR oligomerization in both compartments.4)Further,we demonstrated that HuR oligomerization requires RNA by vitro pull-down experiments,and PARylation can promote HuR to form oligomeric complexes along the target RNA by RNA-EMSA.5)Finally,We proved that HuR oligomerization mediated by PARP1 is essential for stabilizing mRNA through the establishment of target RNA cleavage experiments and the use of W261 E site mutants.These results reveal a new mechanism that PARP1 participates in the regulation of mRNA stability at the post-transcriptional level by promoting the formation of HuR oligomerization.