R2D2 Organizes Small Regulatory RNA Pathways in Drosophila^∇†

Abstract

Drosophila microRNAs (miRNAs) and small interfering RNAs (siRNAs) are generally produced by different Dicer enzymes (Dcr-1 and Dcr-2) and sorted to functionally distinct Argonaute effectors (AGO1 and AGO2). However, there is cross talk between these pathways, as highlighted by the recognition that Drosophila miRNA* strands (the partner strands of mature miRNAs) are generated by Dcr-1 but are preferentially sorted to AGO2. Here, we show that a component of the siRNA loading complex, R2D2, is essential both to load endogenously encoded siRNAs (endo-siRNAs) into AGO2 and to prevent endosiRNAs from binding to AGO1. Northern blot analysis and deep sequencing showed that in the r2d2 mutant, all classes of endo-siRNAs were unable to load AGO2 and instead accumulated in the AGO1 complex. Such redirection was specific to endo-siRNAs and was not observed with miRNA* strands. We observed functional consequences of altered sorting in RNA interference (RNAi) mutants, since endosiRNAs generated from cis-natural antisense transcripts (cis-NAT-siRNA) exhibited evidence for biased maturation as single strands in AGO1 according to thermodynamic asymmetry and a hairpin-derived endo-siRNA formed cleavage-competent complexes with AGO1 upon mutation of r2d2. Finally, we demonstrated a direct role for the R2D2/Dcr-2 heterodimer in sensing central mismatch positions that direct miRNA* strands to AGO2. Together, these data reveal new roles of R2D2 in organizing small RNA networks in Drosophila.Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are conserved families of small regulatory RNAs (11, 60) that operate within related molecular pathways (16,45). In both cases, cytoplasmic Dicer class RNase III enzymes metabolize double-stranded RNA (dsRNA) precursors into small RNA duplexes. These mature into single-stranded RNA associated with an Argonaute (AGO) protein in an RNA-induced silencing complex (RISC), which is guided by the small RNA to target transcripts. Animal miRNAs have an additional preceding processing step in which primary miRNA transcripts are cleaved by the nuclear RNase III enzyme Drosha, yielding ϳ60-to 70-nucleotide (nt) pre-miRNA hairpins that serve as

Derivation and characterization of Dicer- and microRNA-deficient human cells

“…Importantly, miRNAs generated by dDcr1 cleavage are selectively loaded into Drosophila Ago1, while siRNAs generated by dDcr2 cleavage are selectively loaded into Ago2. This latter process is facilitated by a dsRNA-binding protein called R2D2 (Marques et al 2010;Okamura et al 2011;Nishida et al 2013). In contrast, in mammalian cells, miRNAs generated by hDcr are loaded equivalently well into all four Ago proteins (Wang et al 2012).…”

A deeply conserved, noncanonical miRNA hosted by ribosomal DNA

“…However, as the endo-siRNA population is extremely diverse, individual siRNAs are often not well-expressed (thus potentially challenging approaches to assay their depletion in dcr-2 mutants), and immunoprecipitation techniques do not necessarily distinguish intrinsically and peripherally bound species (thus potentially challenging the assessment of all reads in AGO2-IP libraries as bona fide siRNAs). As an alternative strategy, we previously showed that bulk endo-siRNAs are resorted to AGO1 complexes in mutants of the siRNA loading factor R2D2 or the siRNA effector AGO2 (Okamura et al 2011). Such resorting patterns, in which AGO1-IP small RNA reads exhibit relative abundance of r2d2 or ago2 ≫ wild-type .…”

“…First, the proportions of resorted known siRNAs (from transposons and hairpin RNAs) in the AGO1 complexes substantially varied in the four AGO1-IP libraries (from 0.3% in dcr-2 mutant to 25.1% in r2d2 mutant, Supplemental Table S1, Sheet 2). Second, our previous Northern blotting analysis suggested that the levels of abundant miRNA species were not changed in these mutants (Okamura et al 2011). According to this normalization scheme, transposon-derived siRNAs were 27.3 and 36.5 times more abundant in the AGO1-IP libraries from r2d2 and ago2 mutant ovaries, respectively, than the AGO1-IP libraries from wild-type (Supplemental Table S1, Sheet 2).…”

Common and distinct patterns of terminal modifications to mirtrons and canonical microRNAs

“…Although it might be questioned whether such atypically short species are functional, even 19-20-nt miR-317 isomiRs were relatively abundant in AGO1-IP data from ovaries and S2 cells, indicating that they populate endogenous miRISC complexes. Otherwise, AGO1-IP complexes normally select for more conventional ''miRNA-sized'' species (Czech et al 2008;Ghildiyal et al 2010;Okamura et al 2011).…”