The Research On Screening And Identifying Of PprM Target RNAs In Deinococcus Radiodurans

Background: Deinococcus radiodurans is one of the most radiation-resistant organisms known on the earth. It can survive under extremely tough environment of ionizing radiation, ultraviolet light, H2O2, desiccation and some other agents inducing DNA or protein damage. The strong radiation-resistant capability of Deinococcus radiodurans has caught tremendous researchers’ eyes since discovery, while its mechanisms are not clearly understood so far. Previously, several studies have indicated that pprM, a gene in Deinococcus radiodurans, carried out the ability of radiation-resistant. Bioinformatics analyses implied that the PprM protein shares high homology with the cold shock protein and contains multiple RNA-binding domains, which suggested that PprM might as a RNA binding protein and combine with the target RNAs to exert its biological function.Objective: In order to study the interaction between the PprM protein and RNAs, we constructed an over-expression vector of pprM gene and transformed it into Escherchia coli BL21.We isolated and purified the PprM fusion protein, then use RNA pull-down technique and EMSA to find out which RNAs are the targets for PprM protein.Methods: Initially, we constructed a pprM over-expression vector pGEX-6p-1-pprM and transformed it into Escherchia coli BL21. The recombinant protein GST-PprM was expressed after inducting with IPTG and was purified by GST agarose gel. Then coupled the recombinant protein PprM with agarose gel and incubated with the total RNA of Deinococcus radiodurans. We repeatedly eluted the PprM-RNA complexes to gain the PprM binding RNAs, and then connected the specific oligonucleotide linkers to two ends of the target RNAs. The target RNAs were reverse-transcribed to cDNA and cloned into p GEM-T Easy vector to construct a RNA sub-library and identified by sequencing. Finally, the interaction between the target RNAs and PprM were confirmed by EMSA and bioinformatics analysis.Results: We succeeded in constructing the pGEX-6p-1-pprM overexpression vector and without any mutation. We used IPTG to induce the PprM fusion protein expression that purified by GST agarose gel to obtain high purity PprM protein. We successfully screened four target RNAs by RNA pull-Down technology and identified PprM can combine with its own mRNA by EMSA.Conclusions: Successfully constructed the PprM protein over expression vector, and purified the fusion protein PprM. Screened 4 target RNAs which interacted with PprM protein. Identified PprM can combine with its own mRNA5’-UTR.