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

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.     

A C-terminal silencing domain in GW182 is essential for miRNA function

Proteins of the GW182 family are essential for miRNA-mediated gene silencing in animal cells; they interact with Argonaute proteins (AGOs) and are required for both the translational repression and mRNA degradation mediated by miRNAs. To gain insight into the role of the GW182–AGO1 interaction in silencing, we generated protein mutants that do not interact and tested them in complementation assays. We show that silencing of miRNA targets requires the N-terminal domain of GW182, which interacts with AGO1 through multiple glycine–tryptophan (GW)-repeats. Indeed, a GW182 mutant that does not interact with AGO1 cannot rescue silencing in cells depleted of endogenous GW182. Conversely, silencing is impaired by mutations in AGO1 that strongly reduce the interaction with GW182 but not with miRNAs. We further show that a GW182 mutant that does not localize to P-bodies but interacts with AGO1 rescues silencing in GW182-depleted cells, even though in these cells, AGO1 also fails to localize to P-bodies. Finally, we show that in addition to the N-terminal AGO1-binding domain, the middle and C-terminal regions of GW182 (referred to as the bipartite silencing domain) are essential for silencing. Together our results indicate that miRNA silencing in animal cells is mediated by AGO1 in complex with GW182, and that P-body localization is not required for silencing.     

Intertwined pathways for Argonaute-mediated microRNA biogenesis in Drosophila

Although Dicer is essential for general microRNA (miRNA) biogenesis, vertebrate mir-451 is Dicer independent. Instead, its short pre-miRNA hairpin is ‘sliced’ by Ago2, then 3′-resected into mature miRNAs. Here, we show that Drosophila cells and animals generate functional small RNAs from mir-451-type precursors. However, their bulk maturation arrests as Ago-cleaved pre-miRNAs, which mostly associate with the RNAi effector AGO2. Routing of pre-mir-451 hairpins to the miRNA effector AGO1 was inhibited by Dicer-1 and its partner Loqs. Loss of these miRNA factors promoted association of pre-mir-451 with AGO1, which sliced them and permitted maturation into ∼23–26 nt products. The difference was due to the 3′ modification of single-stranded species in AGO2 by Hen1 methyltransferase, whose depletion permitted 3′ trimming of Ago-cleaved pre-miRNAs in AGO2. Surprisingly, Nibbler, a 3′–5′ exoribonuclease that trims ‘long’ mature miRNAs in AGO1, antagonized miR-451 processing. We used an in vitro reconstitution assay to identify a soluble, EDTA-sensitive activity that resects sliced pre-miRNAs in AGO1 complexes. Finally, we use deep sequencing to show that depletion of dicer-1 increases the diversity of small RNAs in AGO1, including some candidate mir-451-like loci. Altogether, we document unexpected aspects of miRNA biogenesis and Ago sorting, and provide insights into maturation of Argonaute-cleaved miRNA substrates.     

HPat a Decapping Activator Interacting with the miRNA Effector Complex

Animal miRNAs commonly mediate mRNA degradation and/or translational repression by binding to their target mRNAs. Key factors for miRNA-mediated mRNA degradation are the components of the miRNA effector complex (AGO1 and GW182) and the general mRNA degradation machinery (deadenylation and decapping enzymes). The CCR4-NOT1 complex required for the deadenylation of target mRNAs is directly recruited to the miRNA effector complex. However, it is unclear whether the following decapping step is only a consequence of deadenylation occurring independent of the miRNA effector complex or e.g. decapping activators can get recruited to the miRNA effector complex. In this study we performed split-affinity purifications in Drosophila cells and provide evidence for the interaction of the decapping activator HPat with the miRNA effector complex. Furthermore, in knockdown analysis of various mRNA degradation factors we demonstrate the importance of NOT1 for this interaction. This suggests that deadenylation and/or the recruitment of NOT1 protein precedes the association of HPat with the miRNA effector complex. Since HPat couples deadenylation and decapping, the recruitment of HPat to the miRNA effector complex provides a mechanism to commit the mRNA target for degradation.     

The GW/WG repeats of Drosophila GW182 function as effector motifs for miRNA-mediated repression

The control of messenger RNA (mRNA) function by micro RNAs (miRNAs) in animal cells requires the GW182 protein. GW182 is recruited to the miRNA repression complex via interaction with Argonaute protein, and functions downstream to repress protein synthesis. Interaction with Argonaute is mediated by GW/WG repeats, which are conserved in many Argonaute-binding proteins involved in RNA interference and miRNA silencing, from fission yeast to mammals. GW182 contains at least three effector domains that function to repress target mRNA. Here, we analyze the functions of the N-terminal GW182 domain in repression and Argonaute1 binding, using tethering and immunoprecipitation assays in Drosophila cultured cells. We demonstrate that its function in repression requires intact GW/WG repeats, but does not involve interaction with the Argonaute1 protein, and is independent of the mRNA polyadenylation status. These results demonstrate a novel role for the GW/WG repeats as effector motifs in miRNA-mediated repression.     

Mechanism of allele-selective inhibition of huntingtin expression by duplex RNAs that target CAG repeats: function through the RNAi pathway

Huntington’s disease is an incurable neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat within one allele of the huntingtin (HTT) gene. Agents that block expression of mutant HTT and preserve expression of wild-type HTT target the cause of the disease and are an alternative for therapy. We have previously demonstrated that mismatch-containing duplex RNAs complementary to the expanded trinucleotide repeat are potent and allele-selective inhibitors of mutant HTT expression, but the mechanism of allele selectivity was not explored. We now report that anti-CAG duplex RNA preferentially recruits argonaute 2 (AGO2) to mutant rather than wild-type HTT mRNA. Efficient inhibition of mutant HTT protein expression requires less AGO2 than needed for inhibiting wild-type expression. In contrast, inhibiting the expression of mutant HTT protein is highly sensitive to reduced expression of GW182 (TNRC6A) and its two paralogs, a protein family associated with miRNA action. Allele-selective inhibition may involve cooperative binding of multiple protein–RNA complexes to the expanded repeat. These data suggest that allele-selective inhibition proceeds through an RNA interference pathway similar to that used by miRNAs and that discrimination between mutant and wild-type alleles of HTT mRNA is highly sensitive to the pool of AGO2 and GW182 family proteins inside cells.     

Defining a new role of GW182 in maintaining miRNA stability

GW182 binds to Argonaute (AGO) proteins and has a central role in miRNA‐mediated gene silencing. Using lentiviral shRNA‐induced GW182 knockdown in HEK293 cells, this study identifies a new role of GW182 in regulating miRNA stability. Stably knocking down GW182 or its paralogue TNRC6B reduces transfected miRNA‐mimic half‐lives. Replenishment of GW182 family proteins, as well as one of its domain Δ12, significantly restores the stability of transfected miRNA‐mimic. GW182 knockdown reduces miRNA secretion via secretory exosomes. Targeted siRNA screening identifies a 3′–5′ exoribonuclease complex responsible for the miRNA degradation only when GW182 is knocked down. Immunoprecipitation further confirms that the presence of GW182 in the RISC complex is critical in protecting Argonaute‐bound miRNA.     

Multiple independent domains of dGW182 function in miRNA-mediated repression in Drosophila

miRNA-mediated repression affects a wide range of biological processes including development and human pathologies. The GW182 protein is a key component of miRNA repression complex, recruited by Argonaute and functioning downstream to repress translation and accelerate mRNA degradation, but little is known about how GW182 proteins act. Using both tethered function and complementation assays, we identify three independent domains of the Drosophila GW182 protein (also termed Gawky) that are sufficient to repress mRNA. Each of these domains also functions independently of poly(A) tails. These results indicate that miRNA-mediated repression is facilitated by multiple domains of GW182.     

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

We have used genome editing to generate inactivating deletion mutations in all three copies of the dicer (hdcr) gene present in the human cell line 293T. As previously shown in murine ES cells lacking Dicer function, hDcr-deficient 293T cells are severely impaired for the production of mature microRNAs (miRNAs). Nevertheless, RNA-induced silencing complexes (RISCs) present in these hDcr-deficient cells are readily programmed by transfected, synthetic miRNA duplexes to repress mRNAs bearing either fully or partially complementary targets, including targets bearing incomplete seed homology to the introduced miRNA. Using these hDcr-deficient 293T cells, we demonstrate that human pre-miRNA processing can be effectively rescued by ectopic expression of the Drosophila Dicer 1 protein, but only in the presence of the PB isoform of Loquacious (Loqs-PB), the fly homolog of the hDcr cofactor TRBP. In contrast, Drosophila Dicer 2, even in the presence of its cofactors Loqs-PD and R2D2, was unable to support human pre-miRNA processing. Interestingly, although ectopic Drosophila Dicer 1/Loqs-PB or hDcr both rescued pre-miRNA processing effectively in these hDcr-deficient cells, there were significant differences in the ratio of the miRNA isoforms that were produced, especially in the case of miR-30 family members, and we also noted differences in the relative expression level of miRNAs vs. passenger strands for a subset of human miRNAs. These data demonstrate that the mechanisms underlying the accurate processing of pre-miRNAs are largely, but not entirely, conserved between mammalian and insect cells.     

Pre-miRNA loop nucleotides control the distinct activities of mir-181a-1 and mir-181c in early T cell development

Background

Mature miRNAs can often be classified into large families, consisting of members with identical seeds (nucleotides 2 through 7 of the mature miRNAs) and highly homologous ∼21-nucleotide (nt) mature miRNA sequences. However, it is unclear whether members of a miRNA gene family, which encode identical or nearly identical mature miRNAs, are functionally interchangeable in vivo.

Methods and Findings

We show that mir-181a-1, but not mir-181c, can promote CD4 and CD8 double-positive (DP) T cell development when ectopically expressed in thymic progenitor cells. The distinct activities of mir-181a-1 and mir-181c are largely determined by their unique pre-miRNA loop nucleotides—not by the one-nucleotide difference in their mature miRNA sequences. Moreover, the activity of mir-181a-1 on DP cell development can be quantitatively influenced by nucleotide changes in its pre-miRNA loop region. We find that both the strength and the functional specificity of miRNA genes can be controlled by the pre-miRNA loop nucleotides. Intriguingly, we note that mutations in the pre-miRNA loop regions affect pre-miRNA and mature miRNA processing, but find no consistent correlation between the effects of pre-miRNA loop mutations on the levels of mature miRNAs and the activities of the mir-181a-1/c genes.

Conclusions

These results demonstrate that pre-miRNA loop nucleotides play a critical role in controlling the activity of miRNA genes and that members of the same miRNA gene families could have evolved to achieve different activities via alterations in their pre-miRNA loop sequences, while maintaining identical or nearly identical mature miRNA sequences.     

Identification of novel microRNA-like-coding sites on the long-stem microRNA precursors in Arabidopsis

Plant microRNA (miRNA) is a crucial regulator of gene expression. It has been reported that more than one miRNA/miRNA* duplex could be produced from a microRNA precursor (pre-miRNA). In this study, we performed a comprehensive search for the novel miRNA candidates on the pre-miRNAs of Arabidopsis. AGO1 enrichment, co-existence of the miRNA*-like coordinates, and unique genome-wide match sites were taken into consideration for candidate screening. As a result, 43 miRNA-like candidates derived from 25 pre-miRNAs were identified. Among these candidates, 31 strong candidates from 22 pre-miRNAs passed all the filtering steps. Interestingly, some of these miRNA-like candidates showed organ-specific expression patterns. After target prediction and degradome sequencing data-based validation, five miRNA candidate–target pairs (ath-miR863-5p.2–AT1G76550.1, ath-miR822.2–AT5G03552.1, ath-miR822.3–AT5G02350.1, sRNA4–AT1G66290.1 and sRNA6–AT1G66310.1) were identified, providing a basis for in-depth functional analysis of these miRNA candidates. These results could update the current understanding of the biogenesis and the action of the plant miRNAs.