Silence Blog

HOT paper: Loqs and R2D2 act sequentially in the siRNA pathway in Drosophila

Two small RNA pathways, namely the small-interfering RNA (siRNA) and microRNA (miRNA) pathway, act ubiquitously in Drosophila somatic tissues (1).  These two pathways differ in the factors required for the biogenesis and function of small RNAs: Dicer-1 (Dcr-1) and its double-stranded RNA (dsRNA)-binding partner, Loquacious (Loqs), generate miRNAs, whereas siRNA-formation requires Dicer-2 (Dcr-2) and the dsRNA-binding protein R2D2 (2,3,4,5,6).

Strikingly, Loqs was also reported to affect siRNA-mediated silencing by an artificially introduced inverted-repeat (IR) transgene targeting the white gene (3). Following the discovery of endogenous siRNAs, the restriction of Loqs to the miRNA pathway was again challenged: Loqs was required for generation of siRNAs derived from endogenous hairpin RNAs, termed structured loci (7,8). However, these hairpins were only partially base-paired (similar to the shorter pre-miRNAs), in contrast to the completely base-paired white-IR transgene (9). It was proposed that Dcr-2 partners with Loqs to generate siRNAs from structured loci, with no dependence on R2D2 (7,8).

Marques et al., in their recent Nature Structural and Molecular Biology paper, followed up on these controversial observations by detailed analysis of the involvement of either R2D2 or Loqs on biogenesis and function of siRNAs derived from different sources (10). High-depth sequencing from loqs mutants confirmed, that the production of miRNAs as well as siRNAs derived from an inverted-repeat transgene requires Loqs. R2D2, however, was only required for normal accumulation of siRNAs, but not miRNAs. They also observed a stronger dependence on Loqs relative to R2D2 for in vitro and in vivo processing of long dsRNA precursors. Moreover, r2d2 mutants failed to assemble a siRNA duplex into an Argonaute complex in vitro, however complex assembly was functional though severely deprived in loqs mutants. Therefore, the authors suggest that the two dsRNA binding proteins act sequentially in the RNAi pathway: Loqs is required along with Dcr-2 for producing siRNAs from dsRNA, whereas R2D2 partners with Dcr-2 to load siRNA duplexes into Ago2.

To further corroborate their conclusion, they show similar results for endogenous siRNAs. Interestingly, they report a dependency for Loqs not only for structured loci-derived siRNAs, confirming previous results (7,8), but also transposons-derived siRNAs. Remarkably, silencing of target mRNAs by endogenous siRNAs is dependent on R2D2, in contrast to previous results observed in S2 cells (7).

Although the disruption of the siRNA pathway in loqs mutants is cogent, the final approval of a hierarchical role for Loqs and R2D2 requires further analysis of Dcr-2-mediated processing using kinetic rather than endpoint assays, possibly in a minimal, reconstituted system. Finally, the effect of loqs mutation on the siRNA pathway could be an indirect effect, even more so, since a physical association between Loqs and Dcr-2 could not be shown.

Megha Ghildiyal and Stefan L. Ameres

References:

1.   Ghildiyal, M. & Zamore, P. D. Small silencing RNAs: an expanding universe. Nat Rev Genet 10, 94-108 (2009).

2.   Lee, Y. S. et al. Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117, 69-81 (2004).

3.   Förstemann, K. et al. Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol 3, e236 (2005).

4.   Saito, K., Ishizuka, A., Siomi, H. & Siomi, M. C. Processing of pre-microRNAs by the Dicer-1-Loquacious complex in Drosophila cells. PLoS Biol 3, e235 (2005).

5.   Jiang, F. et al. Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila. Genes Dev 19, 1674-1679 (2005).

6.   Liu, Q. et al. R2D2, a Bridge Between the Initiation and Effector Steps of the Drosophila RNAi Pathway. Science 301, 1921-1925 (2003).

7.   Czech, B. et al. An endogenous small interfering RNA pathway in Drosophila. Nature 453, 798-802 (2008).

8.   Okamura, K. et al. The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs. Nature 453, 803-806 (2008).

9.   Lee, Y. S. & Carthew, R. W. Making a better RNAi vector for Drosophila: use of intron spacers. Methods 30, 322-329 (2003).

10.   Marques, J. T. et al. Loqs and R2D2 act sequentially in the siRNA pathway in Drosophila. Nature Structural & Molecular Biology (2009).

Posted by Stefan Ameres at 10:30    Comments (0)