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. 2011;6(12):e28548.
doi: 10.1371/journal.pone.0028548. Epub 2011 Dec 6.

The role of Dicer protein partners in the processing of microRNA precursors

Edyta Koscianska  1 , Julia Starega-Roslan, Wlodzimierz J Krzyzosiak
Affiliations

The role of Dicer protein partners in the processing of microRNA precursors

Edyta Koscianska et al. PLoS One. 2011.

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Approaches used to analyze the Dicer step of miRNA biogenesis in cells.
Left: endogenous miRNAs expressed in HeLa cells were analyzed after depletion of AGO2, PACT, TRBP and Dicer with specific siRNAs, and exogenous miRNAs expressed from vector constructs were analyzed after depletion of AGO2, PACT and TRBP. Right: synthetic pre-miRNA precursors were transfected to cells or cleaved by recombinant Dicer, or pri-miRNA encoding vectors were transfected to cells. The results of all analyses were evaluated by northern blotting.
Figure 2
Figure 2. Dicer partners influence endogenous miRNA production.
(A) RT-PCR and western blot analyses of the cellular levels of AGO2, PACT, TRBP and Dicer transcripts and proteins 48 h after the second transfection of HeLa cells with LUC, AGO2, PACT and TRBP siRNAs. An asterisk shown in the case of AGO western blot detection indicates cross-reaction with radixin. (B) Northern blotting detection of miR-21 and pre-miR-21 after transfection with specific siRNAs, as indicated in the figure. M1 denotes size marker, end-labeled 17, 19, 21, 23, and 25-nt oligoribonucleotides. Hybridization to U6 RNA provides a loading control. (C) A bar graph showing quantification of the miRNA and pre-miRNA levels detected by northern blotting as presented in B. (D) Quantitative representations of miRNA length variants obtained from the phosphorimaging analyses shown in B. Signals of the highest intensity in each sample were brought to the same height in order to compare contributions from less intense signals. (E–G) Relevant analyses as in B–D, but performed for miR-16 and pre-miR-16.
Figure 3
Figure 3. Dicer partners influence exogenous miRNA production.
(A) RT-PCR and western blot analyses of cellular levels of AGO2, PACT, TRBP and Dicer transcripts and proteins 72 h after transfection of HeLa cells with LUC, AGO2, PACT and TRBP siRNAs. An asterisk shown in the case of AGO western blot detection indicates cross-reaction with radixin. (B) Northern blotting detection of miR-182 and pre-miR-182 after transfection with specific siRNAs, as indicated in the figure. M1 denotes end-labeled 19, 21, 23, and 25-nt size marker oligoribonucleotides. Hybridization to U6 RNA provides a loading control. (C) Bar graphs showing quantification of the miRNA and pre-miRNA levels detected by northern blotting, as presented in B. (D) Quantitative representations of the miRNA length variants obtained from phosphorimaging analyses from B. Signals of the highest intensity in each sample were brought to the same height in order to compare contributions from less intense signals. (E–H) Relevant analyses as in A–D, but performed for miR-191 and pre-miR-191.
Figure 4
Figure 4. Processing of exogenous pre-miRNA in cells.
(A) Northern blot analysis of RNA from in vitro reaction (line ‘DICER’) and cellular RNA (line in ‘cell’) with probes specific for miRNA derived from the 5′-arm of pre-miR-526b and pre-miR-139 and the 3′-arm of pre-miR-132. A quantitative representation of the cleavage patterns generated in synthetic pre-miRNAs by recombinant Dicer (green) and cellular Dicer (red) is shown below as peaks obtained from the phosphorimaging analysis. The asterisk marks the ∼40-nt intermediate product of one RNase III Dicer domain cleavage. The black bar on the right side marks the miRNA and pre-miRNA fractions; (B) The same as in A, but antisense probes for miR-139 (3′-arm) and miR-132 (5′-arm) were used; (C) Northern blotting analysis with probes detecting miRNA derived from the 3′-arms of precursors after transfection of HeLa cells with vectors encoding pri-miR-137 and pri-miR-206. The bars on the right side mark the miRNA and pre-miRNA fractions. M1 denotes the low molecular weight RNA marker (USB), M2 – end-labeled 23-nt oligoribonucleotide, and M3 – end-labeled 17, 19, 21, 23, 25-nt oligoribonucleotides.
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