Characterization of the TRBP domain required for Dicer interaction and function in RNA interference

Abstract

Background: Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain.

HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs

“…Cell lysates were prepared, separated and transferred for immunoblotting as previously described. 34,83,84 Membranes were blocked for 1 h in 5% nonfat milk and 0.05% Tris-buffered saline (TBS)-Tween and incubated overnight at 4 C with anti-EGFP (Santa Cruz) antibody at a 1/1000 dilution, anti-Myc (Santa Cruz) antibody at a 1/1000 dilution, anti-actin (Chemicon) antibody at a 1/ 10,000 dilution, anti-Dicer349 5 or anti-Dicer (Medimabs, Montreal, QC, CA) at a 1/5000 dilution, anti-TRBPjbx 36 at a 1/500 dilution, anti-Ago2 7C6 (from Dr. T. Hobman) at a 1/5000 dilution, anti-HIV-1 p24 183-H12-5C 83,84 at a 1/1000 dilution, anti-Ad2/5 E1A (Santa Cruz) at a 1/1000 dilution, or anti-GAPDH (Santa Cruz) at a 1/1000 dilution in 5% milk/TBST. After 5 washes in TBST, membranes were incubated with peroxidase-conjugated secondary donkey anti-rabbit antibody (Amersham) for TRBP, Ago2, Dicer and E1A, and sheep antimouse (Amersham) for EGFP, Myc, Actin, p24 and GAPDH at a 1/5000 dilution.…”

“…For RNA-IPs, HeLa cells or HEK 293T cells were transfected with pCMV-Myc or pCMVMyc-TRBP2 and cotransfected with pSIREN-TAR, pSIREN-RRE or pcDNA3-RRE. Cells were lysed and extracts were immunoprecipitated in RNase-free conditions as described 34 using anti-Myc (Santa Cruz) antibodies. One quarter of the immunoprecipitate was used to recover bound proteins by boiling the beads for 10 min and fractionated by 7.5% SDS-PAGE.…”

“…34,83,84 For virus production, HEK 293T cells were transfected with HIV-1 provirus pNL4-3 and supernatants were harvested 48 h post-transfection. Viral content of supernatant was evaluated by performing a reverse-transcriptase assay as described.…”

“…16,32 TAR has been proposed to disrupt RNAi activity by binding TRBP, thereby sequestering it from the RISC. 33 TRBP has been shown to be a crucial, nonredundant component of RNAi, 4,34 so its removal from the RISC would be highly detrimental to RNAi function. However, knockdown of TRBP decreases HIV-1 replication, suggesting that either RNAi does not restrict HIV-1 RNA or that TRBP has other crucial functions for the virus independent of RNAi.…”

Trans-activation Response (TAR) RNA-binding Protein 2 Is a Novel Modulator of Transient Receptor Potential Canonical 4 (TRPC4) Protein

“…The PDZ-binding domain located at the distal C terminus of TRPC4 and TRPC5 (16) binds to the Na ϩ /H ϩ exchange regulatory factor protein, and, in addition to coupling TRPC4 and TRPC5 to other members of the signaling cascade, such as phospholipase C␤ (16) and G protein-coupled receptors (48), Na ϩ /H ϩ exchange regulatory factor protein may indirectly link TRPC4 to the cytoskeleton. Tarbp2 is predominantly present in the cytoplasm, whereas Dicer can be observed in the nucleus and in the cytoplasm (49). The Dicer binding site in Tarpb2 is the 69 aa C4 domain, located at the C-terminal end of Tarbp2 (49).…”