TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing

Abstract

Dicer is a key enzyme involved in RNA interference (RNAi) and microRNA (miRNA) pathways. It is required for biogenesis of miRNAs and small interfering RNAs (siRNAs), and also has a role in the effector steps of RNA silencing. Apart from Argonautes, no proteins are known to associate with Dicer in mammalian cells. In this work, we describe the identification of TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) as a protein partner of human Dicer. We show that TRBP is required for optimal RNA silencing mediated by siRNAs and endogenous miRNAs, and that it facilitates cleavage of pre-miRNA in vitro. TRBP had previously been assigned several functions, including inhibition of the interferon-induced double-stranded RNA-regulated protein kinase PKR and modulation of HIV-1 gene expression by association with TAR. The TRBP-Dicer interaction shown raises interesting questions about the potential interplay between RNAi and interferon-PKR pathways.

Figure 1

TRBP and Dicer co-immunoprecipitation. (A,B) Anti-Dicer antibodies (Abs) pull down endogenous TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) in extracts of human embryonic kidney (HEK)293 cells, and co-immunoprecipitation of Dicer and TRBP is not sensitive to ribonuclease (RNase) treatment (B). mAb, monoclonal antibody; pAb, polyclonal antibody. (C) Dicer–TRBP interaction studied with a HEK293 cell line expressing haemagglutinin (HA)-tagged TRBP2. A HEK293/HA–LacZ cell line was used as a control. The identity of the Abs, some at increasing concentrations (+ through ++++), used for immunoprecipitation (IP) is indicated at the top of the panels. Abs used for western blots are indicated on the right.

Figure 2

TRBP and Dicer co-sedimentation on glycerol gradients. (A,B) Sedimentation of cytoplasmic extracts from P19 cells (A) and human embryonic kidney (HEK)293 cells (B). Gradient fractions were analysed for Dicer and TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) by western blotting and for miR-17 by northern blotting. (C) Activity of fractions analysed in (B), pooled as indicated (top of the panel), in processing 30-bp double-stranded RNA into small interfering RNA.

Figure 3

Interaction of TRBP with Dicer studied with purified proteins (A,B) and in the yeast two-hybrid system (C), and schematic representation of different TRBP transcripts expressed in human cells (D). (A) SDS–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of indicated purified recombinant proteins. MBP, maltose-binding protein. (B) Purified TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) and Dicer interact with each other. Added proteins, antibodies (Abs) and amylose beads are indicated at the top. Abs used for western blotting are shown on the right. HA, haemagglutinin; mAb, monoclonal antibody. (C) Dicer and TRBP interact in a yeast two-hybrid (2H) assay through the carboxy-terminal domain of TRBP. Top: scheme of TRBP2 and its fragments included in 2H constructs. Double-stranded RNA-binding domains (dsRBDs) are shown as grey boxes. Bottom: β-galactosidase staining diagnostic of the interaction between proteins expressed as fusions with either Gal4 DNA activation domain in pGADGH (GAD) or Gal4 DNA binding domain in pGBT9 (GBD). Cyclin T1/Tat and TRBP2/TRBP2 are positive controls. Tat/TRBP2 is a negative control. (D) Scheme of the TRBP gene and its encoded transcripts. Intron positions and exon regions encoding three dsRBDs are indicated. Two alternative transcription starts, and translation initiation and stop codons, are indicated by arrows, circles and triangles, respectively. Skipping of exon 7 in TRBP3 causes translation to terminate in exon 8.

Figure 4

Depletion of TRBP affects pre-microRNA processing in vitro but has no effect on accumulation of mature microRNAs in vivo. (A) Levels of TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) in 293/TRBPkd1/2 and human embryonic kidney (HEK)293T-REx control cell lines after treatment with tetracycline (Tet) for 72 h as indicated. Anti-TRBP antibody was used for western blotting. (B,C) Effect of TRBP knockdown on processing of pre-let-7 RNA in vitro (B) and on accumulation of mature microRNAs (miRNAs) in vivo (C). (B) Processing of pre-let-7 RNA was assayed in extracts from 293/TRBPkd1, 293/TRBPkd2 and 293/control-hairpin cell lines. Phosphorimage of 293/TRBPkd1 (upper panel) and quantification of data for both TRBPkd cell lines (bottom panels) are shown. (C) Total RNA isolated from TRBPkd cell lines, from two independent cultures and control cell lines was analysed by northern blot using probes specific for indicated RNAs. Quantification of data from four independent northern blots showed no significant differences in miR-16 and miR-17 levels between TRBPkd and control cultures. A representative experiment is shown. Similar results were obtained for miR-19B and let-7 miRNAs (data not shown).

Figure 5

Effect of TRBP depletion on RNA silencing. Depletion of TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) decreases the efficiency of RNA interference mediated by the endogenous microRNA miR-17 (A) and by transfected anti-lamin A/C small interfering RNA (B). Cell lines used for analysis are indicated. In (A), activities of Renilla luciferase (RL)-miR-17-Perf reporter in every cell line are expressed in relation to activities of RL-control reporter (set as 100%). Values are means±s.d. of four transfections (^**P<0.01). Similar results were obtained in several independent experiments. Antibodies used for western blotting in (B) are indicated on the right.