title: 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.
authors: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
authors: Ho Young
authors: HY
authors: Lee
authors: Kaihong
authors: K
authors: Zhou
authors: Alison Marie
authors: AM
authors: Smith
authors: Cameron L
authors: CL
authors: Noland
authors: Jennifer A
authors: JA
authors: Doudna
mesh_terms: D060565:Argonaute Proteins / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012323:RNA Processing, Post-Transcriptional; D034741:RNA, Small Interfering / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D043244:Ribonuclease III / Q000378:metabolism*; D000099253:Trans-Activation Responsive RNA-Binding Protein
chemical_list: D060565:Argonaute Proteins; D053487:DEAD-box RNA Helicases; D035683:MicroRNAs; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D043244:Ribonuclease III; D000099253:Trans-Activation Responsive RNA-Binding Protein; C466422:AGO2 protein, human; C104647:PRKRA protein, human; C408087:DICER1 protein, human
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