The Structure And Function Analysis Of Internal Ribosomal Entry Sites Within FET Family MRNA

Transcription factors of FET family plays an important role in the process of gene expression, The human FET proteins are associated with transcription, splicing, microRNA(miRNA) processing, RNA transport, signaling and maintenance of genomic integrity,and possesses RNA- and DNA-binding abilities. There are many reporters have shown that the members of the family is closely related to the tumorigenesis, it is also found that the proteins of FET are overexpressed in various cancers.In this paper the 5’ UTRs of FET mRNAs were analyzed and found that the UTRs are unusually long and GC rich, it was also found that the 5’ UTRs of EWSR1 and FUS have complex secondary structures. All these features are similar with Internal Ribosomal Entry Site(IRES) which can initiate translation with directly recruit ribosomes to the mRNA. When compared to the secondary structures of NRF and XIAP 5’ UTR which have been demonstrated contain IRES element, discovered that secondary structures of EWSR1 and FUS 5’ UTR have great similarities to them. When the 5’ UTRs of FET were cloned into the bicistronic luciferase plasmid, it was found that the translation level of FL which were mediated by EWSR1 and FUS 5’ UTRs were significantly augment.By using the dual luciferase report plasmid without SV40 promoter, it was demonstrated that FUS 5’ UTR contains an IRES element and there was an cryptic promoter in EWSR1 5’ UTR. To further analysis EWSR1 5’ UTR, a series deleted fragments was made and cloned into the dual luciferase report plasmid without SV40 promoter, the results showed that the 5’ UTR has two IRES elements one is between 1-165 bp(IRES A) and another one is located between 225-328 bp(IRES B), at the same time it was also found that the cryptic promoter activity was mainly depended on the sequence between 145-224 bp. These findings will provide a new understanding way and study directions to FUS and EWSR1 expression.To further determine the core IRES regions within FUS and EWSR1 IRES elements, based on their secondary structures a series deletions were made, and it was found that the core region of FUS IRES activity were the ten bases between 21-30 bp and for EWSR1 IRES B the core region was located between 225-255 bp. Against the core region and secondary structure of EWSR1 IRES B, 8 mutations were made and cloned into dual luciferase report plasmid, the results shown that the sequence and secondary structure are all important to it’s IRES activity. And this finding will lay a foundation for further study the mechanism of IRES.To study the expression of EWSR1 and FUS and their IRES activities under cellular stress, the A2780 cells were treated with serum starvation and antineoplastic paclitaxel(PTX) and cis-platinum(DDP), and using the Western Blot to detect the protein levels of EWSR1 and FUS. The results shown that the expression of EWSR1 was extremely stable under those stresses, but those cellular stresses can stimulate not only FUS protein expression distinctly, but also FUS IRES activity, so we conjecture that those stresses induced FUS IRES mediated translation which led to the increase of the protein expression.