Identification Of MRNA Surveillance Factor PELO Interacting Protein

Background: Messenger RNA(mRNA)is a single-stranded RNA molecule,which is complementary to the DNA strand of female parent.According to the gene in the cell as the template,in accordance with the principle of complementary base pairing,generate mRNA after the transcription,mRNA contains the base sequence corresponding to some functional fragments in DNA molecules.mRNA have the ability of coding,and can be used as a direct template for protein biosynthesis.mRNA only accounts for 2%-5% of the total RNA in biological cells,but it has the largest variety and is highly active in metabolism.It is a kind of RNA with the shortest half-life in the body,and can be decay within a few minutes to a few hours after synthesis.mRNA plays a universal role in life activities,among which,in almost all organisms,gene expression regulation and translation are affected by mRNA stability.It has been reported that mRNA secondary structure,promoter region elements,the length of the transcript,RNA sequence play key roles in the regulation of mRNA stability.Changes in mRNA degradation leads to developmental defects and human disease.The mechanism of mRNA degradation also plays an important part in the antiviral defense system.PELO(mRNA Surveillance And Ribosome Rescue Factor)is a well-known mRNA quality Surveillance And Ribosome Rescue Factor,which is homologous gene with DOM34.Studies have revealed that PELO is take part in several significance biological functions,including cell cycle regulation,spermatogenesis,meiosis,and tumorigenesis and migration.It has also been reported that PELO,an e Rf1 homologous,is a key factor in the NGD/NSD surveillance pathway and assists in release abnormal stagnant ribosome.These results indicate that PELO is involved in the regulation of mRNA decay.But,up to now,the specific regulatory mechanism of PELO on mRNA degradation in human body has not been thoroughly understood and needs to be further explored.POLR2A is the largest subunit of human RNA polymerase ?.It has catalytic function and contains the heptopeptide repeat sequence(CTD),which is crucial for the polymerase activity,also known as RPB1.POLR2 A is involved in many physiological processes,including RNA transcription,elongation,suspension,and termination,and is essential for the maintenance of human function.POl R2 A is involved in the genesis and development of tumors in vivo,affects cell senescence,regulates transcriptional coupling events,and participates in the regulation of gene expression.Since POLR2 A plays an indispensable role in life activities,changes in the stability of POLR2 A at mRNA level will lead to dysregulation of physiological activities in cells.Previous studies in our laboratory have suggested that PELO affects the stability of POLR2 A mRNA,and then the scientific question we posed was the identification of the interacting proteins in which PELO regulates POLR2 A mRNA degradation.Methods:(1)PELO reporter gene containing HA protein tag was constructed(HA is at the PELO 5 'end),and the Hela cells were transfected with transient transfection technique.The expression of PELO protein with HA tag was detected by Western blot.(2)The lentiviral packaging system was used to package the plasmid PELO-HA tag-PLVX-TRE3 G,and the Hela cells were infected with the virus.Puromycin and G418 drugs were used to screen the cell lines,and the stable expression of the protein was tested by Western blot after DOX induction.(3)The localization of PELO protein in cells was verified by immunofluorescence assay and nucleoplasmic separation.(4)HA antibody and HBS1 antibody were incubated with whole cell lysate,respectively,to obtain the protein immunoprecipitation complex,and Western blot was used to test the expression of PELO and HBS1 protein in the complex.(5)Interaction protein screening:the HA antibody was co-incubated with whole cell lysate to obtain the protein immunoprecipitation complex,which was analyzed by mass spectrometry and GO analysis after silver staining.Results:(1)PELO reporter gene containing HA protein tag was constructed in PLVX-TRE3 G plasmid(HA is at the PELO 5 'end).Western blot results showed that the PELO protein containing HA protein tag could be overexpressed normally in cells after transfection 48 h.(2)PELO-HA tag overexpression model was successfully constructed in He La cell lines,and the protein expressions of PELO and HA were simultaneously up-regulated in the overexpressed cell lines by Western blot.(3)PELO was distributed in both the nucleus and cytoplasm,but the expression level of PELO was higher in the nucleus before induction and in the cytoplasm after induction.The localization of PELO protein was partially shifted after overexpression.(4)Western blot did not detect the presence of HBS1 protein in the protein immunoprecipitation complex of the HA antibody.Similarly,Western blot did not detect the presence of PELO protein in the protein immunoprecipitation complex of the HBS1 antibody.(5)Silver staining results showed that compared with the control group,the experimental group had different positions of bands,and the efficiency of IP test was higher test by Western blot.The results of mass spectrometry showed that HBS1 was not present in human He La cells,which were mainly ribosomal proteins and ATP-related proteins.The GO analysis indicated that these major interacting proteins were involved in mRNA degradation pathway.Conclusion: The above experimental data suggest that PELO may not interact with HBS1 protein to affect the stability of POLR2 A mRNA in human normal He La cells.PELO may interact with ribosomes in an ATP-dependent manner to regulate the stability of POLR2 A mRNA.