HIV-1 Tat interaction with Dicer: requirement for RNA

Abstract

Dicer is an RNase III which processes two classes of cellular small RNAs: the microRNAs (miRNA) and short interfering RNAs (siRNA). Previously, we observed that over-expressed HIV-1 Tat protein can suppress the processing of small RNAs inside cells. Here, we have investigated the requirements for Tat interaction with Dicer. We report that Tat-Dicer interaction depends on RNA, requires the helicase domain of Dicer, and is independent of Tat's transactivation domain.

Figure 1

Tat co-immunoprecipitation with Dicer requires RNA. A) 293T cells were transfected with pcDNA-Dicer-myc (lane 1) or cotransfected with pcDNA-Dicer-myc and pcDNA-wtTat-flag (lane 2) or Tat point mutants, TatK41A or TatK51A (lane 3 and 4). 48 hours later, cell lysates were immunoprecipitated with anti-myc beads overnight at 4°C. Dicer-immunoprecipitates were assessed by Western blotting using anti-myc (top panel) and co-immunoprecipitated Tat was detected using anti-flag (middle panel). As a control, the amounts of wt Tat and Tat mutants were verified in total cell lysates (lower panel). B) Co-immunoprecipitation analyses of transfected samples after no treatment (lane 1 to 4) or treatment with 50 μg/ml of RNase A (lanes 5 to 8). In addition to immunoblotting for Dicer and Tat, presence of TRBP in the immunoprecipitations was also analyzed.

Figure 2

Dicer's helicase domain is required for co-immunoprecipitating Tat. A) Co-immunoprecipations were performed after transfection of Dicer mutants deleted from the N-terminus progressively to encompass the DEAD domain (ΔDEAD), the helicase domain (ΔHelicase), the Domain of Unknown Function 283 (ΔDUF), and the PAZ domain (ΔPAZ) as schematically illustrated in panel B. Cell lysates (lanes 7 to 12) and immunoprecipitations using anti-flag beads were characterized by immunoblotting using anti-flag (upper panel), anti-Tat (middle panel) or anti-Ago2 (bottom panel). B) Schematic illustration of the Dicer mutants and summary of the co-immunoprecipitation between Dicer and Tat and Dicer and TRBP.

Figure 3

Tat's transactivation domain Tat (1–45) does not pull-down Dicer from cell lysates. A) Purified GST-Tat and four Tat-deletion mutants, described in B, were used for GST pull down assays of cell lysates from myc-Dicer transfected 293T cells. GST, GST-Tat and GST-Tat mutants were first verified by immunoblotting using anti-GST. The pulled-down of Dicer was analyzed by immunoblotting using anti-myc antibody. B) Schematic illustration of Tat mutants and summary of the pull-down results.