Endogenous mouse Dicer is an exclusively cytoplasmic protein

Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse

Unknown Areas of Activity of Human Ribonuclease Dicer: A Putative Deoxyribonuclease Activity

The Dicer ribonuclease plays a crucial role in the biogenesis of small regulatory RNAs (srRNAs) by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. Dicer-generated srRNAs can control gene expression by targeting complementary transcripts and repressing their translation or inducing their cleavage. Human Dicer (hDicer) is a multidomain enzyme comprising a putative helicase domain, a DUF283 domain, platform, a PAZ domain, a connector helix, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Specific, ~20-base pair siRNA or miRNA duplexes with 2 nucleotide (nt) 3’-overhangs are generated by Dicer when an RNA substrate is anchored within the platform-PAZ-connector helix (PPC) region. However, increasing number of reports indicate that in the absence of the PAZ domain, binding of RNA substrates can occur by other Dicer domains. Interestingly, truncated variants of Dicer, lacking the PPC region, have been found to display a DNase activity. Inspired by these findings, we investigated how the lack of the PAZ domain, or the entire PPC region, would influence the cleavage activity of hDicer. Using immunopurified 3xFlag-hDicer produced in human cells and its two variants: one lacking the PAZ domain, and the other lacking the entire PPC region, we show that the PAZ domain deletion variants of hDicer are not able to process a pre-miRNA substrate, a dsRNA with 2-nt 3ʹ-overhangs, and a blunt-ended dsRNA. However, the PAZ deletion variants exhibit both RNase and DNase activity on short single-stranded RNA and DNAs, respectively. Collectively, our results indicate that when the PAZ domain is absent, other hDicer domains may contribute to substrate binding and in this case, non-canonical products can be generated

A New Short Oligonucleotide-Based Strategy for the Precursor-Specific Regulation of microRNA Processing by Dicer

<div><p>The precise regulation of microRNA (miRNA) biogenesis seems to be critically important for the proper functioning of all eukaryotic organisms. Even small changes in the levels of specific miRNAs can initiate pathological processes, including carcinogenesis. Accordingly, there is a great need to develop effective methods for the regulation of miRNA biogenesis and activity. In this study, we focused on the final step of miRNA biogenesis; i.e., miRNA processing by Dicer. To test our hypothesis that RNA molecules can function not only as Dicer substrates but also as Dicer regulators, we previously identified by SELEX a pool of RNA oligomers that bind to human Dicer. We found that certain of these RNA oligomers could selectively inhibit the formation of specific miRNAs. Here, we show that these specific inhibitors can simultaneously bind both Dicer and pre-miRNAs. These bifunctional riboregulators interfere with miRNA maturation by affecting pre-miRNA structure and sequestering Dicer. Based on these observations, we designed a set of short oligomers (12 nucleotides long) that were capable of influencing pre-miRNA processing <i>in vitro,</i> both in reactions involving recombinant human Dicer and in cytosolic extracts. We propose that the same strategy may be used to develop effective and selective regulators to control the production of any miRNA. Overall, our findings indicate that the interactions between pre-miRNAs and other RNAs may form very complex regulatory networks that modulate miRNA biogenesis and consequently gene expression.</p></div

The influence of 12-nucleotide 2′-O-methylated oligomers targeting apical fragments of pre-miRNA hairpins on miRNA production by hDicer.

<p>(<b>A</b>) Radiolabeled pre-miRNAs were incubated with hDicer and 12-nt 2OMe-oligomers. Control reactions lacked the enzyme and oligomer (C−) or the oligomer only (C+). Triangles represent increasing amounts of the indicated 2OMe-oligomers (pre-miRNA:oligomer molar ratios of 1∶1, 1∶10, and 1∶100). The diagrams show the average efficiency of miRNA production based on three independent experiments; error bars represent the standard deviations. (<b>B</b>) Binding of 2OMe-oligomers to the corresponding pre-miRNAs. Radiolabeled oligomers were denatured and renatured alone (−) or in the presence of the corresponding pre-miRNAs (+). The reactions were separated in a native polyacrylamide gel. The positions of the complexes formed by the oligomers and pre-miRNAs are indicated with arrows.</p

Twelve-nucleotide oligomers targeting apical fragments of pre-miRNA hairpins disturb precursor processing by hDicer.

<p>(<b>A</b>) The predicted secondary structures of four tested pre-miRNAs (pre-miR-16-1, pre-miR-21, pre-miR-33a and pre-miR-210). Oligonucleotides are shown as thick, black curves. The calculated melting temperatures of duplexes formed by nucleotides present in the apical fragments of each pre-miRNA and corresponding oligomer are shown below the structures. (<b>B</b>) Radiolabeled pre-miRNAs were incubated with hDicer in the presence of the appropriate 12-nt oligomers. Control reactions lacked the enzyme and oligomer (C−) or the oligomer only (C+). Triangles represent increasing amounts of indicated oligomers (pre-miRNA:oligomer molar ratios of 1∶1, 1∶10, and 1∶100). The diagrams show the average efficiency of miRNA production based on three independent experiments; error bars represent the standard deviations. (<b>C</b>) Binding of the 12-nt oligomers to the corresponding pre-miRNAs. Radiolabeled oligomers were denatured and renatured alone (−) or in the presence of the corresponding pre-miRNAs (+). Reactions were separated in a native polyacrylamide gel. The position of complexes formed by oligomers and pre-miRNAs are indicated with arrows.</p