A hallmark of RNA silencing is a class of approximately 22-nucleotide RNAs that are processed from double-stranded RNA precursors by Dicer. Accurate processing by Dicer is crucial for the functionality of microRNAs (miRNAs). The current model posits that Dicer selects cleavage sites by measuring a set distance from the 3′ overhang of the double-stranded RNA terminus. Here we report that human Dicer anchors not only the 3′ end but also the 5′ end, with the cleavage site determined mainly by the distance (~22 nucleotides) from the 5′ end (5′ counting rule). This cleavage requires a 5′-terminal phosphate group. Further, we identify a novel basic motif (5′ pocket) in human Dicer that recognizes the 5′-phosphorylated end. The 5′ counting rule and the 5′ anchoring residues are conserved in Drosophila Dicer-1, but not in Giardia Dicer. Mutations in the 5′ pocket reduce processing efficiency and alter cleavage sites in vitro. Consistently, miRNA biogenesis is perturbed in vivo when Dicer-null embryonic stem cells are replenished with the 5′-pocket mutant. Thus, 5′-end recognition by Dicer is important for precise and effective biogenesis of miRNAs. Insights from this study should also afford practical benefits to the design of small hairpin RNAs.

Highlights d A short motif in IntS8 mediates association with protein phosphatase 2A (PP2A) d Recruitment of PP2A is necessary for Integrator-mediated gene repression d Integrator-bound PP2A dephosphorylates residues within the RNA Pol II CTD and Spt5

Summary Several terminal uridyltransferases (TUTases) are known to modulate small RNA biogenesis and/or function via diverse mechanisms. Here, we demonstrate that Drosophila splicing-derived pre-miRNAs (mirtrons) are efficiently modified by the previously uncharacterized TUTase, Tailor. Tailor is necessary and sufficient for mirtron hairpin uridylation, and this modification inhibits mirtron biogenesis. Genomewide analyses demonstrate that mirtrons are dominant Tailor substrates, and three features contribute to substrate specificity. First, reprogramming experiments show Tailor preferentially identifies splicing-derived miRNAs. Second, in vitro tests indicate Tailor prefers substrate hairpins over mature miRNAs. Third, Tailor exhibit sequence preference for 3′-terminal AG, a defining mirtron characteristic. Our work supports that Tailor preferentially suppresses biogenesis of mirtrons, an evolutionarily adventitious pre-miRNA substrate class. Moreover, we detect preferential activity of Tailor on 3′-G canonical pre-miRNAs, and specific depletion of such loci from the pool of conserved miRNAs. Thus, Tailor activity may have had collateral impact on shaping populations of canonical miRNAs.

While Slicer activity of Argonaute is central to RNAi, conserved roles of slicing in endogenous regulatory biology are less clear, especially in mammals. Biogenesis of erythroid Dicer-independent mir-451 involves Ago2 catalysis, but mir-451-KO mice do not phenocopy Ago2 catalytic-dead (Ago2-CD) mice, suggesting other needs for slicing. Here, we reveal mir-486 as another dominant erythroid miRNA with atypical biogenesis. While it is Dicer dependent, it requires slicing to eliminate its star strand. Thus, in Ago2-CD conditions, miR-486-5p is functionally inactive due to duplex arrest. Genome-wide analyses reveal miR-486 and miR-451 as the major slicing-dependent miRNAs in the hematopoietic system. Moreover, mir-486-KO mice exhibit erythroid defects, and double knockout of mir-486/451 phenocopies the cell-autonomous effects of Ago2-CD in the hematopoietic system. Finally, we observe that Ago2 is the dominant-expressed Argonaute in maturing erythroblasts, reflecting a specialized environment for processing slicing-dependent miRNAs. Overall, the mammalian hematopoietic system has evolved multiple conserved requirements for Slicer-dependent miRNA biogenesis.

ErbB3 harbors weak kinase activity, but strongly activates downstream phosphatidylinositol 3-kinase/Akt signaling through heterodimerization with and activation by other ErbB receptor tyrosine kinases. We report here that ErbB3 loss in the luminal mammary epithelium of mice impaired Akt and MAPK signaling and reduced luminal cell proliferation and survival. ERBB3 mRNA expression levels were highest in luminal mammary populations and lowest in basal cell/stem cell populations. ErbB3 loss in mammary epithelial cells shifted gene expression patterns toward a mammary basal cell/stem cell signature. ErbB3 depletion-induced gene expression changes were rescued upon activation of Akt and MAPK signaling. Interestingly, proliferation and expansion of the mammary basal epithelium (BE) occurred upon ErbB3 targeting in the luminal epithelium, but not upon its targeting in the BE. Multiple cytokines, including interleukin 6, were induced upon ErbB3 depletion in luminal epithelium cells, which increased growth of BE cells. Taken together, these results suggest that ErbB3 regulates the balance of differentiated breast epithelial cell types by regulating their growth and survival through autocrine-and paracrinesignaling mechanisms.A berrant regulation of the ErbB family of receptor tyrosine kinases (RTKs) and their ligands is common in human cancers (1-4). This family consists of four members: HER1/ErbB1/ EGFR (epidermal growth factor receptor), HER2/ErbB2/Neu, HER3/ErbB3, and HER4/ErbB4. Except for ErbB3, which has weak kinase activity, the ErbB RTKs exhibit dimerization-induced phosphorylation and catalytic activation. In response to ligand binding, ErbBs form homodimers and heterodimers with other ErbB coreceptors. ErbB3 relies on transphosphorylation by heterodimeric partners to induce signal transduction (5-7).ErbB RTKs are required for breast development, although each receptor bears a unique spatiotemporal expression pattern. ErbB2 loss in the mammary epithelium delays ductal elongation during puberty and disorganizes cells within terminal end buds (TEBs) (8-10). EGFR and ErbB4 are not required for mammary ductal development. Rather, EGFR is expressed in the basal epithelium (BE) and in the mammary stroma, and ErbB4 is necessary for milk production (11,12). Although classical knockout of mouse ErbB3 results in embryonic lethality (13), transplant experiments showed that ErbB3 drives growth of the mammary epithelium during puberty (8). Although the mechanism(s) by which ErbB2 and ErbB3 regulate growth of the ductal epithelium are currently unknown, such knowledge will impact our understanding of the earliest events contributing to the formation of ErbB2/HER2-amplified breast cancers, which account for 20-30% of all breast cancers. ErbB3-ErbB2 heterodimers are the most potent oncogenic ErbB-signaling pair due in part to strong ErbB3-induced phosphatidylinositol 3-kinase (PI3K) activation in response to ErbB3 tyrosine phosphorylation at six PI3K interaction motifs (14,15).To understand the role of ErbB3 in mammary gland dev...

While most miRNA knockouts exhibit only subtle defects, a handful of miRNAs are profoundly required for development or physiology. A particularly compelling locus is Drosophila mir-279, which was reported as essential to restrict the emergence of CO2-sensing neurons, to maintain circadian rhythm, and to regulate ovarian border cells. The mir-996 locus is located near mir-279 and bears a similar seed, but they otherwise have distinct, conserved, non-seed sequences, suggesting their evolutionary maintenance for separate functions. We generated single and double deletion mutants of the mir-279 and mir-996 hairpins, and cursory analysis suggested that miR-996 was dispensable. However, discrepancies in the strength of individual mir-279 deletion alleles led us to uncover that all extant mir-279 mutants are deficient for mature miR-996, even though they retain its genomic locus. We therefore engineered a panel of genomic rescue transgenes into the double deletion background, allowing a pure assessment of miR-279 and miR-996 requirements. Surprisingly, detailed analyses of viability, olfactory neuron specification, and circadian rhythm indicate that miR-279 is completely dispensable. Instead, an endogenous supply of either mir-279 or mir-996 suffices for normal development and behavior. Sensor tests of nine key miR-279/996 targets showed their similar regulatory capacities, although transgenic gain-of-function experiments indicate partially distinct activities of these miRNAs that may underlie that co-maintenance in genomes. Altogether, we elucidate the unexpected genetics of this critical miRNA operon, and provide a foundation for their further study. More importantly, these studies demonstrate that multiple, vital, loss-of-function phenotypes can be rescued by endogenous expression of divergent seed family members, highlighting the importance of this miRNA region for in vivo function.

SummaryPostnatal mammary gland development and differentiation occur during puberty and pregnancy. To explore the role of DNA methylation in these processes, we determined the genome-wide DNA methylation and gene expression profiles of CD24+CD61+CD29hi, CD24+CD61+CD29lo, and CD24+CD61−CD29lo cell populations that were previously associated with distinct biological properties at different ages and reproductive stages. We found that pregnancy had the most significant effects on CD24+CD61+CD29hi and CD24+CD61+CD29lo cells, inducing distinct epigenetic states that were maintained through life. Integrated analysis of gene expression, DNA methylation, and histone modification profiles revealed cell-type- and reproductive-stage-specific changes. We identified p27 and TGFβ signaling as key regulators of CD24+CD61+CD29lo cell proliferation, based on their expression patterns and results from mammary gland explant cultures. Our results suggest that relatively minor changes in DNA methylation occur during luminal differentiation compared with the effects of pregnancy on CD24+CD61+CD29hi and CD24+CD61+CD29lo cells.

Distinct mammalian RNA viruses trigger Dicer-mediated production of virus-derived small-interfering RNAs (vsiRNA) and encode unrelated proteins to suppress vsiRNA biogenesis. However, the mechanism and function of the mammalian RNA interference (RNAi) response are poorly understood. Here, we characterized antiviral RNAi in a mouse model of infection with Nodamura virus (NoV), a mosquito-transmissible positive-strand RNA virus encoding a known double-stranded RNA (dsRNA)-binding viral suppressor of RNAi (VSR), the B2 protein. We show that inhibition of NoV RNA replication by antiviral RNAi in mouse embryonic fibroblasts (MEFs) requires Dicer-dependent vsiRNA biogenesis and Argonaute-2 slicer activity. We found that VSR-B2 of NoV enhances viral RNA replication in wild-type but not RNAi-defective MEFs such as Argonaute-2 catalytic-dead MEFs and Dicer or Argonaute-2 knockout MEFs, indicating that VSR-B2 acts mainly by suppressing antiviral RNAi in the differentiated murine cells. Consistently, VSR-B2 expression in MEFs has no detectable effect on the induction of interferon-stimulated genes or the activation of global RNA cleavages by RNase L. Moreover, we demonstrate that NoV infection of adult mice induces production of abundant vsiRNA active to guide RNA slicing by Argonaute-2. Notably, VSR-B2 suppresses the biogenesis of both vsiRNA and the slicing-competent vsiRNA-Argonaute-2 complex without detectable inhibition of Argonaute-2 slicing guided by endogenous microRNA, which dramatically enhances viral load and promotes lethal NoV infection in adult mice either intact or defective in the signaling by type I, II, and III interferons. Together, our findings suggest that the mouse RNAi response confers essential protective antiviral immunity in both the presence and absence of the interferon response. IMPORTANCE Innate immune sensing of viral nucleic acids in mammals triggers potent antiviral responses regulated by interferons known to antagonize the induction of RNA interference (RNAi) by synthetic long double-stranded RNA (dsRNA). Here, we show that Nodamura virus (NoV) infection in adult mice activates processing of the viral dsRNA replicative intermediates into small interfering RNAs (siRNAs) active to guide RNA slicing by Argonaute-2. Genetic studies demonstrate that NoV RNA replication in mouse embryonic fibroblasts is inhibited by the RNAi pathway and enhanced by the B2 viral RNAi suppressor only in RNAi-competent cells. When B2 is rendered nonexpressing or nonfunctional, the resulting mutant viruses become nonpathogenic and are cleared in adult mice either intact or defective in the signaling by type I, II, and III interferons. Our findings suggest that mouse antiviral RNAi is active and necessary for the in vivo defense against viral infection in both the presence and absence of the interferon response.