Study On IRES Element Intracellular Activity In The 5'UTR Of STAT2

As important intracellular transcriptional factors,Signal Transducer and Activator of Transcription(STAT)protein family can have multi-aspect impact upon various physiological processes,such as cellular immunity,proliferation,apoptosis,differentiation etc.A unique member of them,one of the features of STAT2 is its inability to form homodimers,yet it still plays major roles in various physiological processes.The earliest as well as most well known function of STAT2 is to bind with STAT1 and Interferon Regulatory Factor 9(IRF9),result in forming the trimeric complex IFN-Stimulated Gene Factor 3(ISGF3),to promote immune response in cells or organisms.With more intensive studies on STAT2 being conducted,it was discovered that high expression level of STAT2 could promote carcinoma cell proliferation as well as drug resistance.Internal Ribosome Entry Site(IRES)is a sequence that can mediate RNA to be translated in a cap-independent manner,which can help cells to better cope with stressful environments,and its sequence itself can form stable and relatively complex secondary structures with a high GC-content.As illustrated by those studies that STAT2 can promote cell survival during stressful times,together with the fact that the sequence of 5'UTR in STAT2(acquired from Genbank)is GC-rich and can stably form several stem-loop structures,indicate that the5'UTR of STAT2 may possess IRES activity.By utilizing dual-luciferase reporter assays,it is discovered that the 5'UTR of STAT2 can effectively mediate the translation initiation of downstream firefly luciferase.To verify that whether the 5'UTR of STAT2 mediates the translation of downstream reporter gene through IRES or not,the possibilities of cryptic promoter,cryptic splice sites,and read-through or ribosomal shunting have to be ruled out.By constructing the vectors w/o SV40 promoter,the vectors w/ hairpin structures that can obstruct cap-dependent translations,and designing primers to conduct q PCR,the existences of these phenomena have been excluded,thus successfully proved the IRES activity in the 5'UTR of STAT2.Then truncated sequences are constructed according to the secondary structure of 5'UTR in STAT2,and the active central domain is located to nt 33-142(with two stem-loop structures)in the 5'UTR of STAT2 by dual-luciferase reporter assays.So far,a novel mechanism in the translational regulation of STAT is discovered,which will provide those studies on mechanisms and drug designs related to STAT2 with some new ideas.Next,sg RNAs are designed based on where the IRES activity central domain is located,followed by the utilization of CRISPR-Cas9 genome editing tools to construct STAT2 IRES active central domain knockout HEK293 cell lines,and then the homozygous monoclonal knockout cell line is screened out by dilution cloning.During the comparison of the growth curves between knockout and wild-type 293 in normal environment,the growth rate of those knockout ones is discovered to be slightly higher than the wild-type ones,indicating that the knocked-out sequence has affected 293 cell proliferation.Further studies are needed to elucidate the underlying mechanism.