Mechanism Of GRSF1 Interacting Proteins Involved In Up-Regulation Of HTERT Expression By MiR-346

[Objective] MicroRNAs(miRNAs)are highly conserved single-stranded,non-coding RNA molecules of approximately 22 nt that are ubiquitous in eukaryotic cells and generally down-regulate gene expression at the post-transcriptional level.There is increasing evidence that miRNAs can activate transcription or translation.RNA-binding proteins(RBPs)serve as universal regulators of gene expression,affecting the cleavage of mRNA precursors,the transport,localization,turnover and translation of mature mRNA.RBPs can interact directly or indirectly with miRNAs to regulate the expression of specific mRNA synergistically or competitively.We previously found that GRSF1 binds to the specific motif "CCGCAU" of miR-346,and the flanking sequence of miR-346 binds to hTERT 3’UTR to form a GRSF1-miR-346 / hTERT mRNA complex and promotes its recruitment to ribosomes,thereby increasing the expression of hTERT.Herein,we aim to investigate the detailed regulatory molecular mechanisms by which GRSF1 mediates miR-346 binding to the3’UTR of the target gene and upregulates its expression.[Methods] Firstly,we screened the interacting proteins of GRSF1 by Flag affinity purification combined with mass spectrometry.After reviewing a large body of literatures,we initially identified candidate proteins that may be related to GRSF1 function.It was analyzed by immunoprecipitation(Co-IP)and immunofluorescence assays(IFA)combined with laser confocal microscopy whether they interacted with and colocalized with GRSF1.Then we constructed a series of truncated plasmids of KIF11,RPS2,Ago2 and TRIM21 proteins,and confirmed the specific domain of these four proteins binding to GRSF1 by Co-IP assays.The effects of KIF11,RPS2 and TRIM21 on the expression of hTERT were detected by Western blot and RT-qPCR assays.RNA-IP was performed to analyze the binding of KIF11 and hTERT mRNA.The effect of KIF11 on the distribution of hTERT mRNA in ribosomes was detected by sucrose density gradient centrifugation.Western blot and IFA were utilised to detect the effect of GRSF1 on the intracellular distribution of RPS2 and the colocalization of GRSF1 with RPS2 or Ago2.We analyzed the effect ofGRSF1 on the amount of hTERT mRNA loaded on Ago2 by RIP.The effect of TRIM21 on the half-life of GRSF1 was detected by Western blot.We investigated the effect of TRIM21 on the ubiquitination level of GRSF1 by ubiquitination Co-IP.[Results] GRSF1 interacts with KIF11,RPS2,TRIM21,Ago2 and colocalizes with them in the cytoplasm,while GRSF1 and RPS2 are also colocalized in the nucleus.KIF11 and RPS2 promote hTERT expression,while TRIM21 inhibits hTERT expression,and their effect on hTERT expression is dependent on the presence of miR-346 and their interaction with GRSF1.KIF11 binds to hTERT mRNA in GRSF1 dependent manner.KIF11 promotes the transport of hTERT mRNA to polysomes requiring KIF11 motility and microtubule structure integrity.Interfering with the interaction of GRSF1-KIF11 in vivo attenuates the effect of KIF11 on hTERT mRNA transport.GRSF1 up-regulates the expression of RPS2 at the post-transcriptional level and promotes the transport of RPS2 from the nucleus to the cytoplasm.When miR-346 was overexpressed,the colocalization degree of GRSF1 and RPS2 increased,while the colocalization degree of GRSF1 and Ago2 decreased.GRSF1 inhibits the loading of miR-138 and hTERT mRNA on Ago2.TRIM21 relies on its E3 ubiquitin ligase activity to promote the ubiquitination of GRSF1 and decrease its stability.[Conclusion]We found that KIF11 transports the GRSF1-miR-346/hTERT mRNA complex to ribosomes in a GRSF1-dependent manner,thereby facilitating translation of hTERT.GRSF1 selectively facilitates translation of hTERT by up-regulating the expression of RPS2 at the post-transcriptional level and promoting the transport of RPS2 from the nucleus to the cytoplasm.GRSF1 competitively inhibits the assembly of miR-138 and hTERT mRNA into the RISC complex by Ago2,thereby suppressing the down-regulation of hTERT by miR-138.Moreover,TRIM21,as a ubiquitinating enzyme of GRSF1,restrains GRSF1-mediated up-regulation of hTERT by miR-346 by regulating the stability of GRSF1.