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


RESULTS
We show that the TRBP binding site in Dicer is a 165 amino acid (aa) region located between the ATPase and the helicase domains. The binding site in TRBP is a 69 aa domain, called C4, located at the C-terminal end of TRBP. The TRBP1 and TRBP2 isoforms, but not TRBPs lacking the C4 site (TRBPsDeltaC4), co-immunoprecipitated with Dicer. The C4 domain is therefore necessary to bind Dicer, irrespective of the presence of RNA. Immunofluorescence shows that while full-length TRBPs colocalize with Dicer, TRBPsDeltaC4 do not. tarbp2-/- cells, which do not express TRBP, do not support RNA interference (RNAi) mediated by short hairpin or micro RNAs against EGFP. Both TRBPs, but not TRBPsDeltaC4, were able to rescue RNAi function. In human cells with low RNAi activity, addition of TRBP1 or 2, but not TRBPsDeltaC4, rescued RNAi function.


CONCLUSIONS
The mapping of the interaction sites between TRBP and Dicer show unique domains that are required for their binding. Since TRBPsDeltaC4 do not interact or colocalize with Dicer, we suggest that TRBP and Dicer, both dsRBPs, do not interact through bound dsRNA. TRBPs, but not TRBPsDeltaC4, rescue RNAi activity in RNAi-compromised cells, indicating that the binding of Dicer to TRBP is critical for RNAi function.
Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, Québec, Canada. sylvanne.daniels@mail.mcgill.ca
Sylvanne M
SM
Daniels
Carlos E
CE
Melendez-Peña
Robert J
RJ
Scarborough
Aïcha
A
Daher
Helen S
HS
Christensen
Mohamed
M
El Far
Damian F J
DF
Purcell
Sébastien
S
Lainé
Anne
A
Gatignol
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000099253:Trans-Activation Responsive RNA-Binding Protein
D016601:RNA-Binding Proteins; D043244:Ribonuclease III; D000099253:Trans-Activation Responsive RNA-Binding Protein