TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing

Abstract

MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the posttranscriptional level 1 . Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex2 -4. These pre-miRNAs are cleaved by the RNase III Dicer5 -8 to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence9. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein10), which contains three double-stranded, RNAbinding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer-TRBP with Argonaute 2 (Ago2)11 , 12, the catalytic engine of RISC. The physical association of Dicer-TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer-TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer-TRBP complex not only in miRNA processing but also as a platform for RISC assembly.To gain an insight into the components of the miRNA/siRNA processing machinery, we isolated a Dicer-containing complex from human cells. This was accomplished by developing HEK293-derived stable cell lines expressing Dicer tagged with Flag (Flag-Dicer). Flag-Dicer was isolated using affinity chromatography, and the affinity eluate was subjected to SDSpolyacrylamide gel electrophoresis (PAGE) followed by silver staining and western blot analysis.Western blot and mass spectrometric analyses indicated that most polypeptides in the Dicer affinity eluate were products of the proteolytic break down of Dicer (Fig. 1a). However, mass spectroscopy identified a 50-kDa band (six peptide sequences that migrated slightly above the contaminating MEP50 band) corresponding to the human immunodeficiency virus (HIV)-1 transactivating response (TAR) RNA-binding protein (TRBP) 10 . The TRBP gene encodes a protein with three double-stranded RNA-binding domains (dsRBDs). Analysis of the nonredundant protein database by Blast identified proteins with close homology to TRBP in both vertebrates and Drosophila (CG6866) (Fig. 1b) (Fig. 1c). The presence of Dicer was also confirmed by mass spectrometric sequencing. Moreover, additional bands (indicated with an asterisk in Fig. 1c) correspond to SKB1 and MEP50, common contaminants of Flag purification. Although most of TRBP eluted in smaller fractions (32 and beyond; perhaps as a consequence of overexpression), a minor portion of TRBP eluted as a large complex (fractions 16 and 18) not easily visualized by silver sta...

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“…Pre-miRNA hairpin precursor is exported into the cytoplasm by the Exportin-5/Ran-GTP complex 28,29 and is then bound by the RISC Loading Complex (RLC) containing Dicer, TRBP, and Ago2. [30][31][32] Dicer cleaves the pre-miRNA into the mature miRNA, which enters the RISC complex and binds to complementary sequences within the 3 0 -UTR of the target mRNA. 33,34 RISC-bound mRNA transcripts may be suppressed in protein translation, destabilized by deadenylation, or degraded by Ago2 RNase activity.…”

Principles of micro-RNA production and maturation

“…Dicer or its complex interacts with a family of conserved proteins called Argonautes (Bernstein et al, 2001;Hammond et al, 2001;Doi et al, 2003;Tahbaz et al, 2004;Chendrimada et al, 2005;Forstemann et al, 2005;Gregory et al, 2005;Haase et al, 2005;Jiang et al, 2005;Maniataki and Mourelatos, 2005;Saito et al, 2005;Lee et al, 2006). Mature miRNAs are eventually transferred to Argonaute proteins and serve as guides in RNA silencing (Caudy et al, 2002;Hutva´gner and Zamore, 2002;Ishizuka et al, 2002;Mourelatos et al, 2002;Liu et al, 2004;Meister et al, 2004).…”

Direct regulation of microRNA biogenesis and expression by estrogen receptor beta in hormone-responsive breast cancer

“…These molecules, frequently transcribed as polycistronic RNAs, are synthesized in the nucleus by RNA polymerase II or III as long primary transcripts (pri-miRNAs), that are then processed by the class-2 RNase-III Drosha (Han et al, 2004) in B70-nucleotide stem-loop RNAs (pre-miRNAs), that in turn are exported from nucleus to cytoplasm by exportin 5 and Ran-GTP (Kim et al, 2009) and cleaved by Dicer/TRBP endoribonuclease into an imperfect miRNA/miRNA* duplex (Chendrimada et al, 2005). Only one strand of the duplex is finally selected to function as a mature miRNA, whereas the other (passenger) strand is typically degraded (Okamura et al, 2008;Newman and Hammond, 2010).…”