The Mechanism Of TRBP Participating In RISC

RNA interference( RNAi) is a gene-silencing process during which endogenous messenger RNA is destroyed upon introduction of the corresponding double-stranded RNA. dsRNA is processed by the cellular enzyme Dicer into short,21~23 nucleotide dsRNA segments, referred to as small interfering RNA(siRNA), with a two-nucleotide overhang at each 3'end. These siRNA become incorporated into a RNA-induced silencing complex(RISC), where siRNA serves as a guide to identify homologous mRNA for destruction. Biologically, RNAi-related processes are critical for development, heterochromatin formation, and offer cellular protection from virus and transposon proliferation. RNAi has found widespread application as a technique within research laboratories, allowing simple yet effective knockdown of genes of interest. Despite the widespread use of RNAi to knockdown gene function, the RNAi pathway itself remains poorly understood, especially for the formation of RISC complex.Survivin is a member of IAP members which are the important gene families responsible for apoptosis regulation. Survivin gene is localized at chromosome 17 and has 4 exons and 3 introns. The onco-fetal surviving protein is expressed in embryonic organisms and in various malignant tumoers. Localized on chromosome17, the effector protease receptor1(EPR-1)gene encodes a protein with 337 amino acids. The EPR-1 mRNA contains a 1011nt region complementary to surviving mRNA with only 5nt variation and 6nt insertion, which thus provides a natural model for exploring the mechanisms of strand selection.In 2005, the star molecule TRBP looped into view, which participate in mammalian RNA interference. Late 20th century, it is found that TRBP is a double strand RNA binding protein and TRBP has something with the state that HIV virus suppress immunity cells. In mammalian cells, TRBP involves in RNAi indispensably,accompanying with Dicer. TRBP contains three double strand RNA binding domains.The third dsRBD can interact with Dicer. According to the article, TRBP,AGO2 and DCR can interact between each other. Furthmore, in vitro assay, the tertary complex TRBP-AGO2-DCR can process pre-miRNA, recognize guide strand and passenger strand and facilate mature RISC.There are some researches on related factors of siRNA loading on RISC, but no hints on TRBP participating in strand selection has been made. We construct eukarytic expression vector containing full-length, truncated/recombinant or mutate TRBP gene, and harvest a mammalian cell model by RT-PCR and Western blot screening. Then we identify that the expression of foreign TRBP gene in HEK-293 cells transfected with plasmids: pFLAG-CMV4,pFLAG- CMV4 -TRBP,pFLAG-CMV4-TRBP AB,pFLAG-CMV4-TRBP BC,,pFLAG-CMV4- TRBP BC,pFLAG-CMV4-TRBP AC,pFLAG-CMV4-TRBPm. Moreover, how to associate between different form of TRBP and Ago2 or Dicer was found, especially TRBP deleted of C segment cannot associate Dicer or Ago2. Another discovery is optimal transfection dose of shRNA or siRNA in dual lumino reporter gene system. It is demonstrated that Ago2 plays a key role in RNAi through the interference with RISC components, and it also shows some strand selection effect in DLR system.