Screening Specific RNA-binding Proteins And MicroRNAs For Regulating DUSP1 (MKP-1) Gene Expression

In mammalian cells, stimuli like growth factor and environmental stress can trigger multiple signaling pathways. One of these pathways are mitogen-activated protein kinases (MAPKs), which includes extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Activation of MAPKs, involving phosphorylation on threonine 183 and tyrosine 185 residues, leads to activation of multiple downstream effector proteins that cells carry against the biological external stimulus. It was found that MAPKs signaling pathways was involved in process of infection, inflammation, cells apoptosis, tumor pathology, immune response, cell growth and cell differentiation by regulating the function and expression level of related proteins. Constant activation of MAPKs can lead to severe consequences:at the cellular level, it can lead to excessive production of MAPK-regulated genes, excess proliferation of cell, and unscheduled death in cell; at the levels of tissue and organs, it can lead to complications such as autoimmune disease, septic shock, hypertension, and cancer. Therefore MAPKs’intracellular function must be strictly regulated.Dephosphorylation of MAPKs, which is mediated by MKP (MAPK phosphatase), is one of the most effective modes of deactivation of MAPK signaling pathway. At least ten MKPs have been identified in mammalian cells so far. Structurally, all MKPs maintain both a highly conserved Carboxy-terminal catalytic domain and a less conserved amino-terminal domain that combines the MAPKs. The three amino acid residues in highly conserved Carboxy-terminal catalytic domain are essential for enzymatic catalysis. The cysteine residue is required for the nucleophilic attack of the phosphate group on the activated MAPK and it forms a thiol-phosphate intermediate during catalysis. The conserved arginine residue interacts with the phosphate group of the MAPK phosphothreonine or phosphotyrosine which facilitates transition-state stabilization. The aspartic acid residue can enhance catalysis. The two arginine residues in amino-terminal region are crucial in this interaction. MKP-1 (MAPK phosphatase 1) is also termed DUSP1 (dual specificity phosphatases 1). It was the first to be identified in MKP family. DUSP1 electively inactivates all three MAPK families by dephosphorylating them at catalytic tyrosine and threonine residues intracellularly and acts specifically upon p38 MAPK and JNK. So, DUSP1 is an important negative- feedback regulating factor of MAPK signaling pathway.DUSP1 is an immediate-early gene. Activation of MAPKs leads to chromatin remodeling of DUSP1 and facilitates transcription factors including Spl, Sp3, c-Jun, and AP-1 to bind to its promoter and then induces DUSP1 gene transcription. ERK MAP kinase also increases DUSP1 protein stability by phosphorylation. So far, studies are mainly concentrated on transcription level and translational level of DUSP1 gene expression. In recent years, scientists pay more attentions to post-transcriptional regulation, given its rapid and precise control over gene expression. However, little is known about the post-transcription regulatory mechanism.Post-transcriptional regulation is a process regulation after mRNA transcription yet before protein translation. It mainly includes mRNA localization, translational initiation, transcript stability and translational efficiency. Related cis-regulatory elements are mainly found in the UTR (untranslated regions) of transcripts, including 5’-UTR and 3’-UTR. Among them 3’-UTR is more important. Recent studies have shown that RNA binding proteins (RBPs) and microRNAs (miRNAs) can specifically regulate expression of target genes at post-transcriptional level by binding to target mRNA 3’-UTR.RNA binding proteins are highly conservative and widespread during evolution. They specifically bind to target RNA through its RNA binding domain. They directly or indirectly involved in pre-mRNA shearing, editing, transporting and regulating process of stability and translation. A variety of RBPs can specifically bind to target mRNA 3’-UTR ARE (adenine and uridine-rich elements), regulating positively (such as protein HuR) or negatively (such as protein TTP) intracellular expression of the target gene.MiRNAs are small, single-stranded non-coding RNAs. It is highly conservative throughout evolution. Partial complementary pairing of miRNAs with target mRNAs in the 3’-UTR can cause repression in translation and/or degradation of the mRNAs and thus silencing of target gene. So far, over 1000 different kinds of miRNAs have been identified in human cells. Each miRNA have potential to repress expression of many, perhaps hundreds of, target genes. Studies have revealed that miRNAs involves in a variety of life activities, including control of timing in cell development and differentiation, cell apoptosis, cell proliferation, and immune regulation.In summary, screening for RNA binding proteins and miRNAs, which interact with DUSP1 mRNA 3’-UTR, helps us reveal the post-transcriptional regulatory mechanism of gene expression. And this mechanism will help us better understand how MAPKs signal pathway works.Firstly, via bioinformatic analysis of mouse DUSP1 mRNA 3’-UTR, we found it contains three ARE and five potential miRNAs binding sites. Based on result above, we inserted 3’-UTR of mouse DUSP1 mRNA to the downstream of pGL3 luciferase reporter vector. At the same time, we used Renilla reniformis luciferase as internal reference to eliminate the variation in transfection efficiency and cell environments. The result of Dual-Luciferase activity analysis shows that DUSP1 mRNA 3-UTR can significantly reduce the upstream luciferase reporter gene expression, suggesting that it has cis-regulatory elements which can negative regulate gene expression.Based on experiment above, both luciferase expression module from pGL3-Luc-DUSP1-3’-UTR and renilla luciferase expression module from pRL-TK were co-subcloned into pLenti6-TR vector to construct dual luciferase reporter plasmid pLuDP/RL. The recombinant plasmid pLuDP/RL was co-transfected into NIH3T3 cells with both library of siRNAs and miRNA mimics respectively. And then we detect dual luciferase activity, screening for RNA binding proteins and miRNA that bind to DUSP1 mRNA 3’-UTR which can specifically regulate upstream luciferase reporter gene expression. After the screening, we identified 7 proteins including PABPC1, PCBP2, PUM2, UPF3A, ZC3H12D, ZC3HAV1, KSRP and miRNAs including miR-101a/b and numbers of let-7 family. Let-7a/b/c/d/f/i can regulate upstream luciferase reporter gene expression by binding to mouse DUSP1 mRNA 3’-UTR. According to bioinformatic analysis and preliminary experimental verification, we select let-7/miR-98 as a candidate for further study.Firstly, to test potential targeting of DUSP1 mRNA 3’-UTR by miR-98 or let-7, we generated pLuDP-Mut luciferase constructs containing DUSP1 mRNA 3’-UTR with putative let-7 and miR-98 binding sites mutated. Then we transfected this reporter constructs into NIH3T3 cells followed by assessment of luciferase activity after 24 hrs. We also generated let-7 sponge which decoys and decays endogenous let-7 functions. Then co-transfected it into NIH3T3 cells with pLuDP/R and pLuDP-Mut respectively and then move onto assessment of luciferase activity 48 hrs. We co-transfected let-7a/let-7i/miR-98, pLuDP/RL and pLuDP-Mut into NIH3T3 cells respectively, followed by assessment of luciferase activity 48 hrs. Results shown that compared with the control vector (p<0.0l), luciferase activity significantly increased after potential binding sites of let-7 and miR-98 were mutated in DUSP1 mRNA 3’-UTR; The luciferase activity remarkably decreased in pLuDP/RL transfected with let-7a/let-7i/miR-98 respectively (p<0.01). And compared with the control vector, there is no significant change in pLuDP-Mut, of which potential let-7 and miR-98 binding sites were mutated. In addition, luciferase activity significantly increased in pLuDP/RL transfected with let-7 sponge (p<0.01) while no significant change in pLuDP-Mut, compared with the control vector. These results suggest let-7/miR-98 target DUSP1 mRNA 3’-UTR and suppress its upstream luciferase reporter gene expression in NIH3T3 cells.Finally, in order to further analyze whether let-7/miR-98 regulate DUSP1 gene expression, we treated RAW264.7 cells with chemically synthesized miRNA mimics: anti-let-7a, anti-let-7f or anti-miR-98 for 48 hrs respectively and then measured by Real-time PCR and Western blot. No significant change was found in DUSP1 mRNA levels in both control groups and cells treated with anti-let-7a, anti-let-7f or anti-miR-98. But DUSP1 protein expression significantly increased in cells transfected with anti-let-7a, anti-let-7f or anti-miR-98 compared with the control. This results suggests miR-98 and let-7 results in translational suppression of DUSP1 gene at post-transcriptional level.In conclusion, we found that DUSP1 mRNA 3’-UTR has elements which can negatively regulate gene expression. We also identified potential RNA binding proteins and miRNAs which interact with DUSP1 mRNA 3’-UTR by using Dual-Luciferase Reporter Assay System At the same time, we studied biological function of let-7/miR-98 preliminarily. In this study, we had achieved following results:1. In this study we use bioinformatic method to analyze mouse DUSP1 mRNA 3’-UTR. We found that it contains three ARE and five potential binding sites of miRNAs;2. We successfully constructed luciferase reporter gene expression vectors pLuDP and through dual luciferase reporter gene detection system we found that DUSP1 mRNA 3’-UTR could negatively regulate expression of genes;3. The dual luciferase reporter gene expression vectors pLuDP/RL has been successfully constructed. We obtained 7 potential RNA binding proteins and 9 miRNAs which interact with DUSP1 mRNA 3’-UTR by screening via dual luciferase reporter gene detection system;4. We studied biological function of let-7/miR-98 preliminarily and found that let-7/miR-98 target DUSP1 mRNA 3’-UTR and suppress its upstream luciferase reporter gene expression;5. We transfected RAW264.7 cells with anti-let-7a, anti-let-7f or anti-miR-98 respectively for 48 hrs. No significant change in DUSP1 mRNA levels was found, but DUSP1 protein levels significantly increased. These results suggest that miR-98 and let-7 can result in translational suppression of DUSP1 gene at post-transcriptional level.Therefore, study of negative regulatory function of DUSP1 mRNA 3’-UTR and identification of let-7/miR-98 will help us to learn about molecular mechanisms of post-transcriptional regulation for DUSP1, to research MAPKs signal pathway, and to allow us to better understand process of inflammation, stress, allergy, tumor genesis, and abnormal reaction to host caused by MAPKs signal pathway.