Study Of Internal Ribosome Entry Sites In The 5'-UTRs Of STAT Family

Signal transducers and activators of transcription(STATs),a protein family comprised of seven members: STAT1,STAT2,STAT3,STAT4,STAT5 a,STAT5b and STAT6.These proteins have diverse biological functions that include roles in cell differentiation,proliferation,apoptosis and inflammation.Studies have shown that the abnormal expression of STATs implicated in the occurrence and development of various diseases.However,little studies focus on the translation regulation of STAT family mRNAs.By searching the NCBI databases,we found that STAT family mRNAs have usually long 5?-UTRs which can fold into complex and stable secondary stem-loop structures.These characteristics of STAT family mRNAs suggested that their translation might be mediated by IRES(internal ribosomal entry site).Meanwhile,IRES-mediated translation mechanism may be one of causes for STATs stable expression in cells.In this paper,we carried out a functional and structural analysis of STAT IRESs and exploring their binding proteins.These studies will provide a new approach for the regulation of STAT family proteins and the study of cellular IRES elements.Results were shown as followed:1.We employed the classical dual-luciferase reporter gene system to analyze the STAT family 5?-UTRs for the presence of IRES elements.To rule out apparent STAT family IRES activities resulting from cryptic promoter activity,enhanced ribosomal readthrough or splicing event,we performed a serices of expriments and identified an IRES located in STAT1,STAT2 and STAT4 5'-UTR.In addition,to determine whether the relative activities of the STAT family IRES are dependent upon the cell type,we tested their protein expression levels and IRES activities in multiple cancer cell lines.The results showed a positive correlation between STAT1 and STAT4 IRES activities and their protein expression levels.2.To delimit the minimal sequences necessary for STATs IRES activity,nested deletions from the 5?-end and 3?-end of the STAT1,STAT2 and STAT4 5?-UTRs were created according to their predicted secondary structures.For STAT1 IRES element,the 5?-end region between 1 and 232 carries the strong activity and the two regions including 1 to 26 and 94 to 232 are indispensable for its activity.For STAT2 IRES element,a fragment ranging from 31 to 170 was essential for its IRES activity.Interesting,unlike STAT1 IRES,the core active region of STAT4 was located in the 3?-end,the whole region between 101 and 315 was necessary for its IRES activity.Further mutational analysis of the STAT1 and STAT4 5?-UTRs were performed to provide information on the relationship between the structures of the hairpins with STAT1 and STAT4 IRES activity.According to the predicted secondary structures of STAT1 and STAT4 IRES,we constructed 12 plasmids containing STAT1 5?-UTR mutants and 11 plasmids containing STAT4 5?-UTR mutants.The IRES activities of these mutants in Bel7402 and A2780 cell lines were determined by luciferase reporter assay and the results showed that four stem-loops(1GCUGAG6,22UGAUUGG28,61CCUUUU66 and 86CUUUCC91)within 5?-end of STAT1 5?-UTR were crucial to its IRES activity.Moreover,three stem-loops(199AGAAAUGCAAAU210,273GGAC276 and 301UGAGAGA307)within 3?-end of STAT4 5?-UTR were essential for its IRES activity.3.To explore the function of STAT1 and STAT4 IRES under cellular stress conditions.We performed western blot and immunofluorescence to analyze the expression of STAT1 and STAT4.The results revealed that STAT1 and STAT4 proteion synthesis levels were increased in Bel7402 cells during serum-starvation.In addtion,the STAT1 and STAT4 expressions were does dependent in Bel7402 cells treated with PTX.qRT-PCR was performed to determine the status of mRNA of endogenous STAT1 and STAT4 under cellular stress conditions and we found there were no obvious change in STAT1 and STAT4 mRNA levels.These results imply that enhanced STAT1 and STAT4 expressions might occur at rhe translational level.Meanwhile,the IRES activities of STAT1 and STAT4 5?-UTR were significantly increased in Bel7402 cells during serum-starvation or treated with PTX,indicating that STAT1 and STAT4 IRES upregulated endogenpus STAT1 and STAT4 translation levels under cellular stress conditions.4.IRES-mediated translation depends on its RNA structural organization as well as on its binding proteins.For instance,La,YB1,PTB1 and p97 are four important ITAFs,which conjugated to the IRES region of mRNA and induced the conformational change to facilitate the recruitment of ribosome for IRES.Therefore,the interaction of La,YB1,PTB1 and p97 with STAT1 or STAT4 were analyzed in Bel7402 cells by RNA immunoprecipitation(RIP).The results showed that both YB1 and p97 were the binding proteins of STAT1 and STAT4 IRES.However,STAT1 5?-UTR selectively bound PTB1 and STAT4 5?-UTR selectively bound La in Bel7402 cells.Moreover,we found the IRES activities were reduced under conditions where La,YB1,PTB1 or p97 is silenced by siRNA.After analyzing endogenous La,YB1,PTB1 and p97 protein by western blot,we found that YB1 and p97 expression were significantly increased in Bel7402 cells during serum starved condition or treated with PTX.Serun-starvation or PTX also induced relocation of La and PTB1 from nucleus to the cytoplasm in Bel7402 cells,indicating that these four ITAFs positivily regulate the STAT1 and STAT4 IRES activity which might account for the increased IRES mediated STAT1 and STAT4 translation in Bel7402 cells under cellular stress conditions.5.With the comparison of the secondary structures of STAT1,STAT4 and some identified IRES elements,we found that several mRNAs,which have some IRES trans-acting factors in common,had similar structure features.Based on these common short RNA motifs,we predicted,and then identified the mRNAs of four proteins relating with drug resistance contain an IRES element located in their 5?-UTRs.