To demonstrate the synthetic pre-miRNA cleavage by endogenous complexes, Dicer activity from immunoprecipitates and cellular extracts was used, and synthetic precursors were injected into either the nucleus or the cytoplasm of Xenopus oocytes or early embryos. Here, we studied the effect of Dicer protein Trichostatin A partners on premiRNA cleavage efficiency and specificity. Close attention was paid to the length heterogeneity of generated miRNAs 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 premiRNAs and compared cleavage products with those generated in vitro by 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. In this study, we investigated the role of Dicer protein partners in the processing of miRNA precursors. We used RNAi to silence individual Dicer protein partners and analyzed the effects of their inhibition on the endogenous and exogenous miRNA production by high-resolution northern blotting. It has been well documented in the literature that Dicer depletion causes the accumulation of pre-miRNAs and the decrease in the amount of miRNAs. It has also been shown that the level of several pre-miRNAs was not affected by Dicer depletion. In our experimental system, after the depletion of Dicer by RNAi, we observed reduced levels of mature miRNAs and elevated levels of their precursors. Therefore, our observation is in agreement with the majority of previous reports. We also detected the decrease in miRNA amount upon AGO2, TRBP, and PACT depletion. Similar results were reported in the literature and explained by the reduced efficiency of dicing caused by a deficiency of Dicer protein partners. However, upon AGO2, TRBP and PACT depletion we also observed the decrease in the cellular levels of miRNA precursors, which has not been reported so far. As the deficiency of protein partners of Dicer influences the level of Dicer itself, we hypothesize that the pre-miRNA decrease may result from the decreased cellular levels of the functional RLC and a rapid degradation of pre-miRNAs that did not enter the processing complex. The fact that the reduction of TRBP protein level reduces also the level of Dicer protein is in good accordance with the results of others, suggesting that the lack of TRBP decreases Dicer stability and/or affects the assembly of the Dicer complex. Similarly, upon PACT depletion the fall in the Dicer level was observed. It is not surprising that the knockdown of TRBP and PACT causes similar effects, as these proteins can interact with Dicer in a similar manner and can act redundantly to some extent. As far as AGO2 depletion is concerned, the observed decrease in premiRNAs’ level is a novel finding of this study. Earlier, it has been shown that AGO2, which also has a PAZ domain, can bind and cleave pre-miRNAs. It was also proposed that AGO2 may facilitate the access of pre-miRNAs to Dicer.