The Role Of TRBP In The RNA-induced Silencing Complex

TRBP consists of 366 amino acid residues,and it belongs to the family of dsRNA binding proteins ,with three dsRBDs and a KR-helix motif within the second dsRBD that mediates RNA binding,The third C-terminal basic domain mainly mediates interaction between proteins . TRBP was first isolated as an HIV-1 trans-activation response (TAR) RNA binding protein that enhances virus expression in 1991, and it has many biological functions,including controlling the generating and development of spermatozoon,repressing activation of PKR,regulating HIV-1 gene expression,participating in RNAi process and so on.TRBP is a strong PKR inhibitor by direct binding through its dsRBDs and by dsRNA sequestration. TRBP also has a direct activity on translation independent of PKR but dependent on a structured RNA.TRBP promotes HIV-1 expression and replicates,HIV-1 replicates poorly in astrocytes because of a heightened PKR response, which mediates poor translation of the viral structural proteins. TRBP prevents PKR activation, restores the production of viral proteins and consequently HIV replication. Reserch provides an additional mechanistic explanation for the low HIV replication in astrocytic cells and demonstrates the key role of TRBP in virus translation by counteracting the antiviral immunity mediated by PKR.TRBP,Ago2 and Dicer all function in processing of miRNA and RNAi process. TRBP appears as the bridge between dsRNA and Dicer for Ago2 recruitment. Further research shows a decrease in mature miRNA production and a loss of siRNA function either after TRBP or after Dicer depletion. These data suggest that TRBP recruits Ago2 to Dicer and that it couples the initiation and the execution steps of RNAi. TRBP interacts with Dicer, member of RNaseⅢfamily, and binds dsRNA, then binds Ago2 protein with slicing activity of dsDNA, thus the three of them formRISC complex. One strand of dsRNA is submissed to Ago2 protein, and that brings on generation of siRNA and miRNA. However, which domain of TRBP that plays a role in RNAi, as well aswhich domain interacts with Ago2 have not yet been fully clarified.In order to further discuss interaction between TRBP and Ago2, we use RT-PCR to amplify whole TRBP gene, and constructe truncated / reconstructed gene TAB, TBC, TAC, which containing only two dsRNA-binding domain of the gene.There genes were cloned to eukaryotic expressing vector pFlag-CMV4, The sequences of genes were completely correct as confirmed by DNA sequencing, which were named pFlag-CMV4-TRBP,pFlag-CMV4-TAB,pFlag-CMV4-TAC and pFlag-CMV4-TBC.After the four genes were transiently transfected into HeLa cells, the expression of the aimed genes were detected by Western blot. The plasmids were stably transfected into human cervical carcinoma HeLa cells. We detected the interaction of the whole molecule or truncated/reconstructed TRBP, Ago2 and Dicer through immunoprecipitation, and the results show the whole molecule or truncated/reconstructed TRBP have interaction with Ago2 and Dicer molecule. On the other hand, we confirmed that TRBP has a low expression in glioma cell lines by real-time quantitative PCR and Western blot. Dual-luciferase report assay system showed that, glioma cell lines U251, C6, U87, and SHG44 with low TRBP expression reduced the role of RNA interference. Similarly, siRNA againstβ-Actin were transiently transfected into HeLa cells and U251 cells, we found that the result suggest that the effect of RNA interference is reduced in U251 cells.In summary, we have constructed a full-lengthed TRBP and truncated TRBP TAB, TAC, TAC eukaryotic expression vectors, and preliminary tested its functions, and lay the foundation for further study of the mechanism of its function.