The RNA-Binding Protein KSRP Promotes The Biogenesis Of MIR-150

Background: MiRNAs(miRNAs) are a class of short, endogenously-initiated non-coding RNAs that post-transcriptionally control gene expression by either translational repression or mRNA degradation. MiRNA genes are transcribed in most cases by RNA polymerase II into primary miRNA transcripts(pri-miRNA). After transcription the primary miRNA(pri-miRNA) is cleaved by a complex called the microprocessor that contains DROSHA and DGCR8(DiGeorge critical region 8) as well as additional proteins. DROSHA is an RNase III enzyme and DGCR8 is a protein that is essential for miRNA processing(also known as Pasha). The cleaved transcript, 60-nucleotide long, is referred to as precursor miRNA(pre-miRNA). After nuclear processing, the pre-miRNA is exported to the cytoplasm by Exportin-5 in a complex with Ran-GTP61. Once the premiRNA is in the cytoplasm, the RNase III protein Dicer cleaves off the loop of the pre-miRNA and generates a roughly 22-nucleotide miRNA duplex. The functional strand of the mature miRNA is loaded together with Argonaute(Ago2) proteins into RISC complex. In principle, the miRNA duplex could give rise to two different mature miRNAs. However, in a similar manner to siRNA duplexes, only one strand is usually incorporated into RISC and guides the complex to target mRNAs, leading to translational repression or mRNA degradation. It is evident that miRNAs are playing significant roles in various organisms including developmental timing as well as cell proliferation, differentiation, apoptosis and tumorigenesis. The abnormal expression of MiRNAs lead to disorder of body functions and even disease and tumor formation. Thus MiRNAs has also become popular in the biology research over the years.RNAs in cells are associated with RNA-binding proteins(RBPs) to form ribonucleoprotein(RNP) complexes which are important. The RNA-binding proteins(RBPs) affect the structure and interactions of RNAs, playing critical roles in their biogenesis, stability, function, transport and cellular localization. Recent studies have found RBPs may be not only affect its stability of mRNA, can also affect the biogenesis of MiRNAs. Considering that KSRP could promote the biogenesis of a subset of miRNAs and our laboratory mainly research the effects of miRNAs on erythroid differentiation. we will screen the miRNAs and KSRP whose expression are up regulated simultaneously through the Chip analysis in the erythroid differentiation, and we found that KSRP and miR-150 are up regulated simultaneously. Therefore, our research will start with KSRP and miR-150.Objective: We mainly study the role of RNA binding protein KSRP in miR-150 processing.Methods: In our "RBPDB” site predicting, KSRP is one of the RBPs, who could be combined with the primary sequence of miR-150. We hypothesized that KSRP can contribute to the processing of miR-150.Then we transfected the over-expression plasmid of KSRP to K562 cells. The cells were cultured for 24 h and harvested. Then, we extracted the RNA and proteins of harvested cells, and determined KSRP over-expression by real- time PCR and western blot methods. Afterwards,we designed primers of the primary,precusor and mature sequence of miRNAs for the experimental group,positive control group and the negative control group,which were used to detect the expression of primary, precusor, and mature.Then we collected the cells transfected with KSRP expression plasmid after 24 h, extracted the total RNA and detected the expression of primary,precusor and mature of endogenous miR- 150 by real-time quantitative PCR(real- time PCR) after over expressing KSRP, let- 7a and miR- 23 b as positive and negative controls. We found KSRP played an important role in the processing regulation of miR-150. In order to further validate the influence of KSRP on the processing regulation of miR-150, we built a GFP expression plasmids that were inserted into the 3 'UTR of GFP with miRNA primary sequence of texperimental group,positive control group and negative control group.Then we co-intefected the KSRP expression plasmid and the GFP expression plasmid into 293 TN cells and detected the change of GFP fluorescence intensity after 24 h.Next, we used the western blot method to detect the protein levels of GFP. These results also verified the impact of KSRP on miR-150 processing and mature. In order to verify whether KSRP played roles in miR-150 processing by combining with miR-150, we used RNA-IP experiment to ensure the interaction between KSRP and miR-150.Results: Compared with the control group, the mRNA and protein levels of KSRP were significantly higher that indicated the success of KSRP over expression. Then we tested the expression of primary, precusor and mature of endogenous miRNA after over-expressing KSRP. Compared with control group, the primary expression of miR-150 was decreased while the expression of precusor and mature were increased. Compared with the control group, the let-7a primary expression was decreased while the expression of precusor and mature were increased. Compared with the control group, the expression of primary, precusor, mature of miR-23 b were changed unconspicuously. These phenomenons together illustrated KSRP could promote the processing and mature of miR-150. Then the result was verified further by fluorescence imaging. Compared with the blank control group, the GFP fluorescence intensity was abate in the experimental group, the GFP fluorescence intensity was also abate in the positive control group, but the negative control group had no significant change. These results further illustrated the role of KSRP in miR-150 processing. Afterwards, we confirmed KSRP could interact with miR-150 by RNA – IP experiment.Conclusion: 1. KSRP could promote the processing of endogenous miR-150.2. KSRP can be combined with miR-150.3. KSRP promotes the processing and mature of miR-150 by combined with miR-150...