Differential roles of human Dicer-binding proteins TRBP and PACT in small RNA processing

Abstract

During RNA interference and related gene regulatory pathways, the endonuclease Dicer cleaves precursor RNA molecules to produce microRNAs (miRNAs) and short interfering RNAs (siRNAs). Human cells encode a single Dicer enzyme that can associate with two different double-stranded RNA (dsRNA)-binding proteins, protein activator of PKR (PACT) and trans-activation response RNA-binding protein (TRBP). However, the functional redundancy or differentiation of PACT and TRBP in miRNA and siRNA biogenesis is not well understood. Using a reconstituted system, we show here that PACT and TRBP have distinct effects on Dicer-mediated dsRNA processing. In particular, we found that PACT in complex with Dicer inhibits the processing of pre-siRNA substrates when compared with Dicer and a Dicer-TRBP complex. In addition, PACT and TRBP show non-redundant effects on the production of different-sized miRNAs (isomiRs), which in turn alter target-binding specificities. Experiments using chimeric versions of PACT and TRBP suggest that the two N-terminal RNA-binding domains of each protein confer the observed differences in dsRNA substrate recognition and processing behavior of Dicer-dsRNA-binding protein complexes. These results support the conclusion that in humans, Dicer-associated dsRNA-binding proteins are important regulatory factors that contribute both substrate and cleavage specificity during miRNA and siRNA production.

Figure 1.

TRBP and PACT increase RNA binding of Dicer as a complex with Dicer. (A) Domain architectures of TRBP and PACT. Each consists of three dsRNA-binding domains. (B) dsRNA binding by dsRBPs and the Dicer–dsRBP heterodimers. Dicer alone does not bind to the dsRNA, but Dicer–dsRBP complexes bind to dsRNA. The gel shifts by Dicer–dsRBP complexes and by each dsRBP were distinct showing that dsRBPs help Dicer binding to dsRNA as Dicer–dsRBP complexes. dsRNA A (0.5 μM) and 0, 0.5 or 1 μM protein/complex in 20 µl of binding buffer [20 mM HEPES (pH 7.5), 300 mM KCl, 5% glycerol, 1 mM TCEP] were incubated on ice for 45 min before loading to 6% Native PAGE. The gel was stained with SYBR-Gold.

Figure 2.

TRBP and PACT affect pre-miRNA and pre-siRNA processing by Dicer in non-redundant fashion. (A) Dicer–TRBP (D/T) and Dicer–PACT (D/P) are not redundant in producing different-sized miRNAs in pre-miR-200a and pre-miR-34c processing. Assays were done in a single turnover condition with 10-fold excess amount of protein/complex to RNA. RNA substrates were labeled by ³²P phosphate at 5′ end, which is marked with an asterisk. [RNA] = 5 nM and [Dicer]/[Dicer–TRBP]/[Dicer–PACT] = 50 nM were incubated in reaction buffer for 60 min (see ‘Materials and Methods’ section) and quenched with 1.2 volumes of 2-fold formamide dye. Samples were boiled at 70°C for 10 min before loading to 12% denaturing PAGE. After drying, the gel was exposed to a phosphoscreen. (B) Dicer–PACT processes pre-siRNA (dsRNA W1) much less efficiently than Dicer and Dicer–TRBP in a single turnover condition with 10-fold excess amount of protein/complex to RNA. dsRNA W1 was labeled with ³²P phosphate at 5′ end of the bottom strand, which is marked with asterisk. [RNA] = 5 nM and [Dicer]/[Dicer–TRBP]/[Dicer–PACT] = 50 nM were incubated in reaction buffer. The reactions were quenched at each time point and the following steps were the same as described in (A). (C) Dicer, Dicer–TRBP and Dicer–PACT processing kinetics for pre-miRNAs (pre-let-7a and pre-miR-34c) and pre-siRNAs (dsRNA W1 and dsRNA W2). Dicer–PACT processes pre-siRNA much slower than Dicer and Dicer–TRBP, while processing pre-let-7a and pre-miR-34c as efficiently as Dicer and Dicer–TRBP. A single turnover reaction condition with 10-fold excess amount of protein/complex to RNA was used for kinetic assays as in (A). The quenched samples were boiled and loaded onto 12% denaturing gel. After drying and exposing the gel to phosphoscreen, precursor substrate and product miRNA or siRNA bands were quantified by ImageQuant software (GE Healthcare life sciences). % Cleavage was calculated by 100 × (product miRNA counts)/(total counts − sum of substrate and product RNA counts).

Figure 3.

TRBP and PACT retain their effects on pre-miRNA and pre-siRNA processing by Dicer in Ago2 containing complexes: Dicer–Ago2–TRBP and Dicer–Ago2–PACT. (A) Dicer–Ago2–TRBP (D/A/T) and Dicer–Ago2–PACT (D/A/P) produce the same species of product miRNAs when processing pre-miR-200a and pre-miR-34c as Dicer–TRBP (D/T) and Dicer–PACT (D/P), respectively. Assays were done in a single turnover condition with 10-fold excess amount of protein/complex. [RNA] = 5 nM and [D/A/T] or [D/A/P] = 50 nM were incubated in the reaction buffer and the reaction was quenched after 60 min by adding 1.2 volume of 2-fold formamide dye. The following steps were the same as described in Figure 2A. (B) Dicer–Ago2–PACT (D/A/P) shows extremely low efficiency in pre-siRNA processing compared with Dicer–Ago2–TRBP (D/A/T) while processing pre-let-7a and pre-miR-34c are as fast as Dicer–Ago2–TRBP (D/A/T) as shown in Dicer–PACT and Dicer–TRBP in Figure 2B. For dsRNA W1 processing gel, each reaction has been quenched at the indicated time point and the following steps were the same as described in Figure 2B. For kinetic graphs, pre-let-7a, pre-miR-34c and dsRNA W1 were processed by Dicer–Ago2–PACT (D/A/P) and Dicer–Ago2–TRBP (D/A/T) in a single turnover condition with 10-fold excess amount of protein complex as in (A). The following steps were the same as described in Figure 2C.

Figure 4.

Mechanistic studies of the effects of dsRBPs on Dicer by swapping domain and linker between TRBP and PACT. (A) Construct design for TRBP and PACT chimeric proteins swapping the third domain (T12P3 and P12T3) and linker region between domain 1 and domain 2 (TRBP-PL and PACT-TL). (B) Dicer–T12P3 (D-T12P3) and Dicer–P12T3 (D-P12T3) produce highly similar product miRNAs as Dicer–TRBP (D/T) and Dicer–PACT (D/P), respectively, in processing pre-miR-200a. Dicer–P12T3 processes pre-siRNA (dsRNA W1) much less efficiently than Dicer and Dicer–TRBP as in Figure 2C, which mimics Dicer–PACT processing (Figure 2C). For pre-miR-200a processing gel, each reaction has been quenched after 60 min by adding 1.2 volume of 2-fold formamide dye. The following steps were as same as in Figures 2A and 3A. For kinetic graphs, pre-let-7a, pre-miR-34c and dsRNA W1 were processed by Dicer–P12T3 in a single turnover condition with 10-fold excess amount of protein complex. [RNA] = 5 nM and [Dicer–P12T3] = 50 nM were incubated in the reaction buffer and the reaction was quenched at each time point by adding 1.2 volume of 2-fold formamide dye. The following steps were the same as described in Figures 2C and 3B. (C) Dicer–TRBP-PL and Dicer–PACT-TL process pre-miR-200a producing similar isomiR products to Dicer–TRBP and Dicer–PACT, respectively. Bar graphs show relative isomiR (1nt longer miR)/miR ratio in pre-miR-200a processing by Dicer–dsRBPs (Dicer–TRBP and Dicer–PACT) and Dicer-chimeric dsRBPs (Dicer–T12P3, Dicer–P12T3, Dicer–TRBP-PL and Dicer–PACT-TL). Error bar represents Standard Deviation (SD). (D) Dicer–PACT-TL processes pre-siRNA (dsRNA W1) much less efficiently than Dicer–TRBP-PL, while both Dicer–PACT-TL and Dicer–TRBP-PL processes pre-let-7a and pre-miR-34c efficiently. pre-let-7a, pre-miR-34c and dsRNA W1 were processed by Dicer–PACT-TL and Dicer–TRBP-PL in a single turnover condition with 10-fold excess amount of protein complex. [RNA] = 5 nM and [Dicer–TRBP-PL]/ [Dicer–PACT-TL] = 50 nM were incubated in the reaction buffer and the reaction was quenched at each time point by adding 1.2 volume of 2-fold formamide dye. The following steps were the same as described in Figures 2C and 3B.

Figure 5.

A mechanistic model showing how TRBP and PACT differentiate Dicer processing. The two N-terminal dsRBDs of TRBP and PACT bind to dsRNA substrates and recruit them to Dicer–dsRBP complexes, where cleavage occurs with dsRBPs bound to the dsRNA substrates. The two N-terminal dsRBDs of TRBP and PACT affect dsRNA structure and orientation on Dicer differently, altering Dicer processing.