Abstract
One of the cellular functions of the ribonuclease Dicer is to process microRNA precursors (pre-miRNAs) into mature microRNAs (miRNAs). Human Dicer performs this function in cooperation with its protein partners, AGO2, PACT and TRBP. The exact role of these accessory proteins in Dicer activity is still poorly understood. In this study, we used the northern blotting technique to investigate pre-miRNA cleavage efficiency and specificity after depletion of AGO2, PACT and TRBP by RNAi. The results showed that the inhibition of either Dicer protein partner substantially affected not only miRNA levels but also pre-miRNA levels, and it had a rather minor effect on the specificity of Dicer cleavage. The analysis of the Dicer cleavage products generated in vitro revealed the presence of a cleavage intermediate when pre-miRNA 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.
Highlights
The ribonuclease Dicer cleaves double-stranded RNA into small interfering RNA and microRNA precursors into microRNA
We evaluated the levels of both endogenous and exogenous miRNAs after the depletion of AGO2, PACT, TAR RNA-binding protein (TRBP) and Dicer by RNAi
We investigated the role of Dicer protein partners in the processing of miRNA precursors
Summary
The ribonuclease Dicer cleaves double-stranded RNA (dsRNA) into small interfering RNA (siRNA) and microRNA precursors (pre-miRNA) into microRNA (miRNA). 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 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 the TRBP gene caused defects in the processing of miRNA precursors [10]. The loss of PACT expression was reported to influence the biogenesis of miRNAs [7]
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