Dicer and TRBP interact in vivo and in vitro
We raised monoclonal antibodies (mAbs) against human Dicer (
supplementary Fig S1 online). The mAbs 33, 73 and 83, which effectively immunoprecipitate Dicer from extracts of different cultured cells (data not shown), were used to ide.jpgy proteins associated with Dicer in human embryonic kidney (HEK)293 cells. Proteins retained with either mAbs 33/73/83 or anti‐Myc mAb, used as a control, were separated using two‐dimensional gel electrophoresis, and spots enriched in Dicer immunoprecipitates were processed for mass spectrometry analysis. One protein reproducibly co‐purified with Dicer was ide.jpgied as TRBP, a protein containing three dsRBDs (
supplementary Fig S1 online). Members of the Argonautes family were also among the selected proteins, as were some others, but the reproducibility and significance of their interactions with Dicer were not further investigated (data not shown).
To validate the Dicer–TRBP interaction, we performed co‐immunoprecipitation experiments using either extracts from human HEK293 cells or purified proteins. Two anti‐Dicer antibodies (Abs), mAb 73 and polyclonal Ab 347, but not the control mAb isotypic with mAb 73, immunoprecipitated endogenous TRBP that was present in HEK293 cells, as shown by western blotting with anti‐TRBP Abs (
Fig 1A; several forms of TRBP, with apparent molecular masses of 43–46 kDa, are expressed in human cells (see below)). Treatment with RNases digesting both double‐ and single‐stranded RNAs did not eliminate the association (
Fig 1B), indicating that the interaction is not mediated by RNA. As immunoprecipitating anti‐TRBP Abs were not available, we generated a stable HEK293 cell line, HEK293/HA–TRPB2, expressing the haemagglutinin (HA)‐tagged version of the best‐studied isoform of TRBP, TRBP2. Co‐immunoprecipitation experiments performed with the HEK293/HA–TRPB2 extract and either anti‐HA or anti‐Dicer Abs showed that each Ab was able to pull down the partner protein (
Fig 1C). Further indication that TRBP and Dicer form part of the same complex was provided by gradient sedimentation experiments. Analysis of cytoplasmic extracts prepared from either human HEK293 or mouse teratoma P19 cells showed that Dicer and TRBP, or their mouse counterparts, co‐sediment in a region corresponding to a molecular mass of ∼250 kDa (
Fig 2A,B). Notably, miR‐17, an abundant miRNA in HEK293 cells, was also enriched in this region, as was the activity of processing a 30‐bp dsRNA to siRNA (
Fig 2B,C). Taken together, the data indicate that Dicer and TRBP interact with each other in mammalian cells.
To find out whether the Dicer–TRBP interaction was direct, we purified both proteins, as recombinant fusions with His
6 and a maltose‐binding protein (MBP) from insect cells and
Escherichia coli, respectively (
Fig 3A). The proteins were incubated together and applied either to Protein G–Sepharose beads coated with different Abs or to amylose beads. Dicer mAb 73, but not control anti‐HA mAb, effectively pulled down TRBP2. Likewise, TRBP2 retained on amylose beads pulled down Dicer (
Fig 3B). The low efficiency of the latter pull‐down could be the result of a sterical hindrance caused by the MBP tag or owing to the propensity of TRBP to form homodimers (see below). To eliminate the possibility that proteins co‐purifying with either Dicer or MBP–TRBP2 preparations are involved in binding, we studied the Dicer–TRBP interaction in the yeast two‐hybrid (2H) assay (
Fig 3C). As the budding yeast does not encode TRBP or Dicer homologues, any interaction detected in this system would probably result from direct binding. Plasmids encoding full‐length TRBP2, or different regions thereof, fused to the Gal4 DNA‐binding domain, and a plasmid encoding Dicer appended to the Gal4 activation domain, as well as several control plasmids, were transformed into yeast. We detected interactions between Dicer and TRBP2 and all its mutants encompassing amino acids 228–366. This region of TRBP includes the dsRBD C domain, suggesting that this domain mediates the interaction (see Conclusions). We also detected TRBP2 interacting with itself, which was consistent with its ability to homodimerize (
Daher et al, 2001). Taken together, our results indicate that Dicer and TRBP interact directly with each other.
The 45 kDa TRBP2 consists of three dsRBDs. Another previously described TRBP isoform, TRBP1, differs from TRBP2 at the amino terminus (
Bannwarth et al, 2001; see
Fig 3D). By complementary DNA cloning and by inspecting EST databases, we ide.jpgied another TRBP splice variant, potentially encoding a TRBP3 isoform, which would miss the C‐terminal dsRBD (
Fig 3D). Interestingly, one of the three isoforms of Loqs, the probable
Drosophila homologue of TRBP, is also devoid of the C‐terminal dsRBD (
Förstemann et al, 2005). The biological function of individual TRBP variants remains unknown. The alignment of TRBP2 with dsRBD Dicer protein partners from other species and with a TRBP‐related mammalian protein PACT is shown in
supplementary Fig S3A online. High sequence conservation of the C‐terminal dsRBD in Loqs, TRBP2 and PACT (
supplementary Fig S3B online) suggests that this domain has a function distinct from two other dsRBDs (see below).
TRBP is required for miRNA and siRNA silencing
To assess the potential role of TRBP in RNAi and miRNA pathways, we generated stable HEK293T‐REx cell lines, in which the expression of TRBP is knocked down by RNAi. In cell lines 293/TRBPkd1 and 293/TRBPkd2, the expression of stably integrated short hairpins targeting different regions in TRBP messenger RNA is controlled by a pol III promoter with tetracycline (Tet) operator sites. Real‐time PCR and western analyses indicated that TRBP expression was about fivefold lower at both mRNA and protein levels. In either cell line, partial decrease of the protein had already occurred in the absence of Tet induction, indicating some leakiness of the system (
Fig 4A;
supplementary Fig S2 online). TRBP depletion had no appreciable effect on cell growth (data not shown).
We compared the miRNA‐precursor‐processing activity of extracts prepared from different cell lines (
Fig 4B). Extracts from either TRBPkd cell line processed pre‐let‐7 RNA less efficiently than extracts from control cells. The decrease in activity was not due to destabilization of Dicer, as its level was similar in control and TRBPkd cells (
Fig 5B). Despite extracts from TRBPkd cells being deficient in pre‐miRNA processing, steady levels of several miRNAs in these cells were not significantly different from control HEK293 cells, and there was no apparent accumulation of pre‐miRNAs (
Fig 4C; data not shown). Notably, as in the case of TRBP, depletion of Loqs in
Drosophila S2 cells had no principal effect on mature miRNA levels although extracts of Loqs knockdown cells were deficient in pre‐miRNA processing. However, in contrast to TRBP, depletion of Loqs resulted in accumulation of pre‐miRNAs in S2 cells (
Förstemann et al, 2005;
Saito et al, 2005).
We used the miRNP‐mediated mRNA‐reporter‐cleavage assay to find out whether depletion of TRBP had an effect on the activity of endogenous miRNPs in HEK293 cells. TRBPkd and control cells were transiently transfected with constructs expressing either control
Renilla luciferase (RL) reporter mRNA or a reporter harbouring the site perfectly complementary to miR‐17 in its 3′ untranslated region. In control cells, insertion of the miR‐17 site repressed RL expression by ∼80%. However, in TRBPkd cells, the miRNA‐mediated inhibition was about threefold less pronounced (
Fig 5A), indicating that TRBP is required for either the assembly or functioning of miRNPs.
To investigate whether TRBP has a role in the RNAi reaction mediated by exogenous siRNA, we determined the efficiency of the lamin A/C RNAi in TRBPkd and various control cells. The siRNA treatment had a strong effect on lamin A/C level in parental HEK293T‐REx cells or cells stably expressing a control hairpin. However, lamin A/C depletion was largely abolished in TRBPkd cells. Similar suppression of the lamin A/C knockdown was observed in a HEK293 cell line in which Ago2 was depleted by expression of a specific hairpin (
Fig 5B).
Taken together, our data indicate that TRBP is primarily required for the assembly and/or functioning of si‐ or mi‐RISCs in mammalian cells, but it may also facilitate the cleavage of pre‐miRNAs by Dicer. The apparent discrepancy between the effect of TRBP knockdown on pre‐miRNA processing in cells and cell extracts is readily explained by incomplete depletion of the protein, allowing for the manifestation of processing deficiency
in vitro but not
in vivo. It is worth noting that, as in the case of
Drosophila Loqs (
Förstemann et al, 2005;
Saito et al, 2005) but in contrast to R2D2 (
Liu et al, 2003), depletion of TRBP did not lead to appreciable destabilization of Dicer (
Fig 5B).