Molecular mechanisms that funnel RNA precursors into endogenous small-interfering RNA and microRNA biogenesis pathways in Drosophila

Abstract

In Drosophila, three types of endogenous small RNAs-microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and endogenous small-interfering RNAs (endo-siRNAs or esiRNAs)-function as triggers in RNA silencing. Although piRNAs are produced independently of Dicer, miRNA and esiRNA biogenesis pathways require Dicer1 and Dicer2, respectively. Recent studies have shown that among the four isoforms of Loquacious (Loqs), Loqs-PB and Loqs-PD are involved in miRNA and esiRNA processing pathways, respectively. However, how these Loqs isoforms function in their respective small RNA biogenesis pathways remains elusive. Here, we show that Loqs-PD associates specifically with Dicer2 through its C-terminal domain. The Dicer2-Loqs-PD complex contains R2D2, another known Dicer2 partner, and excises both exogenous siRNAs and esiRNAs from their corresponding precursors in vitro. However, Loqs-PD, but not R2D2, enhanced Dicer2 activity. The Dicer2-Loqs-PD complex processes esiRNA precursor hairpins with long stems, which results in the production of AGO2-associated small RNAs. Interestingly, however, small RNAs derived from terminal hairpins of esiRNA precursors are loaded onto AGO1; thus, they are classified as a new subset of miRNAs. These results suggest that the precursor RNA structure determines the biogenesis mechanism of esiRNAs and miRNAs, thereby implicating hairpin structures with long stems as intermediates in the evolution of Drosophila miRNA.

FIGURE 1.

Interaction of Loqs isoforms with Dicer1 and Dicer2. (A) The Loqs-containing complexes were immunoprecipitated from S2 cells using anti-Loqs antibodies and the proteins contained in the immunoprecipitates were visualized by silver staining. The protein bands corresponding to Loqs-PA, Loqs-PB, and Loqs-PC/Loqs-PD are indicated. Two proteins coimmunopurified with Loqs were identified as Dicer1 and Dicer2 by mass spectrometry analyses. (n.i.) Nonimmune IgG used as a negative control. (B) Western blot analyses using anti-Dicer1 and anti-Dicer2 antibodies confirmed that the immunoprecipitates obtained using anti-Loqs antibodies (panel A) contain both Dicer proteins, along with all Loqs isoforms. (C) Loqs-PA and Loqs-PB interact with Dicer1, whereas Loqs-PD interacts with Dicer2. Each Loqs isoform was expressed in S2 cells, and the anti-myc immunoprecipitated complexes were probed with anti-Dicer1 and anti-Dicer2 antibodies. (h.c.) Heavy chain of anti-c-myc antibody. (D) Deletion mutant analysis of Loqs-PD to determine the domain necessary for interacting with Dicer2. Various deletion mutants of Loqs-PD (m1–m5) were expressed in S2 cells, and their interactions with endogenous Dicer2 were examined. The Loqs-PD-m4 mutant interacted with Dicer2 as efficiently as wt Loqs-PD. The dsRNA-binding domains (light gray boxes) and C-terminal region (dark gray boxes) found exclusively in Loqs-PD but not in other Loqs isoforms, respectively, are indicated (upper diagram). The lower panel shows immunoblotting results of the protein–protein interaction assays. EGFP was used as a negative control.

FIGURE 2.

Small RNA excision activities of the Dicer2–Loqs-PD complex immunopurified from S2 cells. (A) esiRNAs excised from the precursor (pre-sl; the nucleotide sequence and a possible structural formation are shown at the top; the structural formation was predicted by RNA & DNA folding applications; http://mfold.bioinfo.rpi.edu/) were found to be loaded onto AGO2, but not onto AGO1 in S2 cell lysates. (Lysate −) esiRNA precursors incubated without S2 lysate, (lysate +) esiRNA precursors incubated with S2 lysate. (Lower panels) Western blot results showing the specificity of immunoprecipitation. (B) The Dicer2–Loqs-PD complex was capable of excising esiRNAs from the precursor. Loqs-containing complexes immunopurified from S2 cells using anti-Loqs antibodies (anti-Loqs) were employed as positive controls. (C) The Dicer2–Loqs-PD complex failed to excise let-7 from the precursor (pre-let-7), whereas the Loqs-PA and Loqs-PB complexes specifically containing Dicer1 were able to process the precursor. (D) The Dicer2–Loqs-PD complex was also able to excise exo-siRNAs from the precursors (EGFP dsRNAs). (E) Western blot analysis using anti-R2D2 antibodies showed that the Dicer2–Loqs-PD complex also contained the R2D2 protein.

FIGURE 3.

Loqs-PD, but not R2D2, enhanced the Dicer2 esiRNA processing activity in vitro. (A) Coomassie brilliant blue staining of GST-tagged, recombinant Loqs-PD and R2D2 proteins produced in and purified from E. coli. (B) Flag-tagged Dicer2 protein expressed in S2 cells following transfection was affinity-purified with an anti-Flag antibody in a buffer containing 1 M NaCl. Western blot analysis revealed that the Dicer2 fraction isolated under such harsh conditions contained only traces of R2D2 and Loqs proteins. (C) In vitro esiRNA processing assays using the purified Dicer2 obtained in B. While Dicer2 alone did not excise esiRNAs from the precursor (pre-esiRNA; lane 3, Fig. 2A), addition of GST-Loqs-PD (lane 5), but not of GST (lane 4) and GST-R2D2 (lane 6), increased the esiRNA production activity of Dicer2 to the same extent as that of Flag-Dicer2 immunoprecipitated from S2 cells with an anti-Flag antibody (lane 2). GST-Loqs-PD alone did not show any esiRNA processing activity (lane 8). These results indicate that Loqs-PD appears to enhance the ability of Dicer2 to process esiRNAs from their precursors by associating with Dicer2. (D) Both GST-tagged Loqs-PD and R2D2, but not GST itself, were able to bind with Flag-Dicer2 purified from S2 cells under a high-salt condition as in Figure 4B. Note that only the full-length proteins (black arrowheads) seemed to be bound with Dicer2. (*) Heavy chains of anti-Flag antibody, (**) light chains of anti-Flag antibody. (E) GST-Loqs-PD (lane 5) but not GST-R2D2 (lane 6) enhances the ability of Dicer2 to process long dsRNAs into exo-siRNAs.

FIGURE 4.

esiRNA processing using pre-sl deletion mutants. (A) The nucleotide sequences and possible structural formation of various pre-sl deletion mutants (pre-sl-M1 to pre-sl-M5) are shown. Pre-sl-M5 contains the loop of pre-let-7 (gray) instead of the pre-sl loop. (B) esiRNA processing was performed with pre-sl as in Figure 2B. The anti-Loqs immunoprecipitates and both Dicer1–Loqs-PB and Dicer2–Loqs-PD complexes immunoprecipitated with an anti-myc antibody, as in Figure 2B, were utilized as the enzymatic sources. The myc-EGFP complex was employed as a negative control. (C) esiRNA processing with pre-sl-M1, as in B. (D) esiRNA processing with pre-sl-M2, as in B. (E) esiRNA processing with pre-sl-M3, as in B. (F) pre-sl-M4 processing, as in B. Note that the precursor was now processed by myc-Loqs-PB but not by the myc-Loqs-PD complex. (G) pre-sl-M5 processing, as in B.

FIGURE 5.

Small RNAs (esiRNA-sl-4 and esiRNA-sl-4as) arising from the region most proximal to the loop in the hairpin esiRNA precursor are loaded predominantly onto AGO1 in S2 cells. esiRNA-sl-1 was loaded onto AGO2 as expected. RNA pools immunoisolated with AGO1 and AGO2 from S2 cells were probed with DNA oligos for esiRNA-sl-1, esiRNA-sl-4, and esiRNA-sl-4as.

FIGURE 6.

A model for esiRNA, exo-siRNA, and miRNA biogenesis pathways in Drosophila. The Dicer1–Loqs-PB (and the Dicer1–Loqs-PA) complex processes miRNA precursors, whereas the Dicer2–Loqs-PD complex processes esiRNA precursors. The Dicer2–Loqs-PD complex also contains R2D2 and thus processes long dsRNAs into siRNAs. The shorter esiRNA precursor intermediates, which are structurally similar to miRNA precursors, are processed by the Dicer1–Loqs-PB complex; this process gives rise to semiRNAs, a new subset of miRNAs.