, siRNA and miRNA pathways have been viewed as being biochemically compartmentalized. However, the boundary between these pathways has been blurred by recent observations that, depending on the configuration of their precursors, miRNAs (and possibly siRNAs) can be partitioned between Ago1 and Ago2 (Forstemann et al., 2007
; Tomari et al., 2007
). Moreover, Loquacious plays roles both in miRNA biogenesis and in the production of some endo-siRNAs (Czech et al., 2008
; Okamura et al., 2008
). Intimate connections between siRNA and miRNA pathways are also suggested by the observation that knockdown of Ago2 leads to more pronounced silencing by miRNAs (), possibly by increasing the access of Ago1 to miRNAs or other limiting components that were previously bound by Ago2.
Analysis of miRNA and siRNA screens shows extensive overlap between genes that impact these pathways. Suppression of many candidates reduces the efficiency of the target small RNA pathways, indicating that those genes might be components of siRNA- or miRNA-mediated responses. Silencing of a roughly equal number of genes increases silencing, indicating that they encode negative regulators of small RNA pathways. These so-called “enhancer of RNAi” phenotypes might indicate attractive targets for genetic manipulation or small molecule inhibitors that could increase the activity of RNAi in either experimental or therapeutic settings. It is worth noting that our analysis of steady-state levels of small RNAs upon candidate knockdown revealed that for some, enhanced silencing is correlated with increased levels of the small RNA silencing trigger, as is the case for the miRNA pathway candidates such as CG32721, mule, TH1 or flfl (). In contrast, while knocking down CG2807 led to markedly enhanced silencing by the miRNA mimetic CXCR4, the steady-state levels of both CXCR4 and miR-2b significantly decreased. While these effects on the small RNA pathways could be indirect, these observations suggest that some of these negative regulators of RNAi are primarily involved in the biogenesis and/or stability of the small RNA silencing trigger, while others are implicated in the downstream effector steps.
Each pathway was uniquely or differentially affected by a number of genes (). For example, knockdown of one class of genes (caf1
and genes encoding ribosomal proteins L5, L21, L22 and S15) led to decreased silencing by exogenous siRNAs, but enhanced silencing by endo-siRNAs. Suppression of such a class of genes might enhance the production or loading of endo-siRNAs into RISC, thereby depleting the pool available for products of exogenously introduced dsRNAs, a model that has been previously proposed for some loci in C. elegans
(Duchaine et al., 2006
). Knockdown of another group of genes (hsc70-4
) decreased silencing by miRNAs but enhanced silencing by endo-siRNAs. A number of possibilities, including effects on small RNA sorting might account for these observations.
We validated Bel as a bona fide
component of the RNAi pathway. Bel most likely functions at step(s) downstream of siRNA processing and loading, as neither steady state levels of esi-2.1 nor the levels of Ago2-bound esi-2.1 are affected by Bel knockdown (Figs. S2 and S3
). Interestingly, Ago1 and Bantam are also present in the Bel immunoprecipitate, consistent with the co-fractionation of Bel with miRISC (). Thus, Bel may also participate in the miRNA pathway. While none of the bel
dsRNAs met the scoring criteria in the miRNA assay, they did trend consistently (Table S1
Ago1 and Bantam were present in a number of ribosomal protein immunoprecipitates, and the association between Ago1 and these ribosomal proteins was abolished by RNase treatment (). These observations are consistent with the notion that miRISC associates with the translation machinery. Both protein and RNA components of the siRISC are also present in these immunoprecipitates and that knockdown of a number of ribosomal proteins consistently leads to enhanced silencing by endo-siRNAs (Table S1
). Thus, the integrity and function of the translational machinery as a whole may be impacting the small RNA pathways.
In summary, our comparative genome-wide screens (Table S5
) generate a rich resource for further study of the three Argonaute-dependent small RNA regulatory pathways in Drosophila
. These studies not only point to extensive overlap and interplay among small RNA directed silencing machineries in flies but also highlight specific players in each of the three pathways.