The contribution of Dicer protein partners to either the efficiency or the specificity of miRNA biogenesis is important but is still unclear and poorly understood

The contribution of Dicer protein partners to either the efficiency or the specificity of miRNA biogenesis is important but is still unclear and poorly understood. was processed by recombinant Dicer alone. This intermediate was not observed during pre-miRNA cleavage by endogenous Dicer. We demonstrate that AGO2, PACT and TRBP were required for the efficient functioning of Dicer in cells, and we suggest that one of the roles of these proteins is to assure better synchronization of cleavages triggered by two RNase III domains of Dicer. == Introduction == The ribonuclease Dicer cleaves double-stranded RNA (dsRNA) into small interfering RNA (siRNA) and microRNA precursors (pre-miRNA) into microRNA (miRNA). Human Dicer belongs to the RNase III class and contains an N-terminal DExH-box RNA helicase-like domain, a domain originally termed the domain of unknown function (DUF283), a PAZ domain, two RNase III domains, and a double-stranded RNA-binding domain (dsRBD)[1]. The dsRNA processing center of Dicer is formed through intramolecular dimerization of two RNase III domains functioning together to cleave phosphodiester bonds on opposite strands of a dsRNA substrate[2]. The RNase IIIa domain cleaves the 3-arm of pre-miRNA and the RNase IIIb domain cleaves its 5-arm, and both domains must exhibit their activity to generate a miRNA-miRNA* duplex. In human cells, Dicer does not act alone, but in cooperation with protein partners, such as members of the AGO family[3],[4], HIV-1 TAR RNA-binding protein (TRBP)[5],[6], a protein activator of PKR (PACT)[7],[8]and possibly other accessory proteins. The contribution of Dicer protein partners to either the efficiency or the specificity of miRNA biogenesis is important but is still unclear BOC-D-FMK and poorly understood. It has been shown that Dicer, AGO2 and TRBP are necessary components in the formation of the RISC-loading BOC-D-FMK complex (RLC) and the effective production of short RNAs[3][5],[9]. In one study, the depletion of TRBP affected pre-miRNA processingin vitro, but it had no effect on the accumulation of pre-miRNAs and mature miRNAs in cells[6]. In another study, the knockdown of TRBP in human cells was shown to result in lower levels of miRNAs, which suggested that the lack of TRBP decreased Dicer stability[5]. A similar destabilization of Dicer resulting from an impairment of TRBP was reported in the case of human carcinomas, in which frameshift mutations in theTRBPgene caused defects in the processing of miRNA precursors[10]. TRBP and PACT interact with each other and associate with Dicer to facilitate the cleavage of dsRNA[8]. The loss of PACT expression was also reported to influence the biogenesis of miRNAs[7]. Numerous approaches have been used to investigate how Dicer cleaves its pre-miRNA substrates, bothin vitroand in cells. In the simplest system, synthetic miRNA precursors were cleaved by recombinant Dicer[2],[4],[11][14]. The results of these studies showed that the enzyme typically generates heterogeneous products from dsRNAs[15]and pre-miRNAs[2],[11],[13],[14]. The influence of the pre-miRNA structure on the length of the Dicer cleavage products has recently been demonstrated[14]. Two components of the RLC complex, Dicer and TRBP or Dicer and AGO2, were used to analyze cleavages in synthetic pre-miRNAs[5],[16]. Also, the pre-miRNA cleavages by Dicer present within the complete RLC reconstituted from recombinant Dicer, TRBP and AGO2 proteins were analyzed[4]. To demonstrate the synthetic pre-miRNA cleavage by endogenous complexes, Dicer activity from immunoprecipitates[3],[9],[17]and cellular extracts[11],[13],[18]was used, and synthetic precursors were injected into either the nucleus or the cytoplasm ofXenopusoocytes or early embryos[19]. Here, we studied the effect of Dicer protein partners on pre-miRNA cleavage efficiency and specificity. Close attention Rabbit Polyclonal to LDLRAD2 was paid to the length heterogeneity of generated miRNAs BOC-D-FMK after depletion of AGO2, PACT, TRBP and Dicer by RNAi. We showed that the Dicer protein partners affect both miRNA and pre-miRNA levels and have a minor effect on the specificity of Dicer cleavage. We also transfected HeLa cells with synthetic pre-miRNAs and compared cleavage products with those generatedin vitroby recombinant Dicer. The results revealed the appearance of a cleavage intermediate product when pre-miRNA was handled by Dicer alone and the absence of such an intermediate when exogenous pre-miRNA was processed by the endogenous Dicer complex. This suggests the role of Dicer’s protein partners in the synchronization of cleavages triggered by RNase III domains. == Results == Dicer associates with several protein partners in cells, i.e., AGO2, PACT and TRBP..