IL-17-secreting CD4+ T cells are critically involved in inflammatory immune responses. Development of these cells is promoted in vivo and in vitro by IL-23 or TGFβ1 plus IL-6. Despite growing interest in this inflammatory Th subset, little is known about the transcription factors that are required for their development. We demonstrate that Stat3 is required for programming the TGFβ1 plus IL-6 and IL-23-stimulated IL-17-secreting phenotype, as well as for RORγt expression in TGFβ1 plus IL-6-primed cells. Moreover, retroviral transduction of a constitutively active Stat3 into differentiating T cell cultures enhances IL-17 production from these cells. We further show that Stat4 is partially required for the development of IL-23-, but not TGFβ1 plus IL-6-primed IL-17-secreting cells, and is absolutely required for IL-17 production in response to IL-23 plus IL-18. The requirements for Stat3 and Stat4 in the development of these IL-17-secreting subsets reveal additional mechanisms in Th cell fate decisions during the generation of proinflammatory cell types.

MicroRNAs (miRNAs) regulate gene expression by guiding Argonaute proteins to specific target mRNA sequences. Identification of bona fide miRNA target sites in animals is made challenging by uncertainties regarding the base-pairing requirements between miRNA and target as well as the location of functional binding sites within mRNAs. Here we present the results of a comprehensive strategy aimed at isolating endogenous mRNA target sequences bound by the Argonaute protein ALG-1 in C. elegans. Using cross-linking and ALG-1 immunoprecipitation coupled with highthroughput sequencing (CLIP-seq), we identified extensive ALG-1 interactions with specific 3′ untranslated region (UTR) and coding exon sequences and discovered features that distinguish miRNA complex binding sites in 3′ UTRs from those in other genic regions. Furthermore, our analyses revealed a striking enrichment of Argonaute binding sites in genes important for miRNA function, suggesting an autoregulatory role that may confer robustness to the miRNA pathway. miRNAs function as ~22-nucleotide (nt) RNAs that target messenger RNAs (mRNAs) for degradation or translational repression 1, 2. A single miRNA can potentially repress hundreds of genes by binding with partial sequence complementarity to mRNAs3 , 4. By combinatorial regulation of thousands of genes, the miRNA pathway critically influences many developmental programs as well as cellular homeostasis, the disruption of which leads to human disease. Thus, an outstanding challenge has been to distinguish biologically relevant miRNA-target interactions. To date, identification of miRNA target sites has been dependent largely on computational methods that have limited capability for predicting specific and physiologically relevant targets. Addressing this need, several studies have reported biochemical approaches to isolate targets by immunoprecipitation of miRNA effector complexes containing miRNA-mRNA duplexes [5][6][7][8][9][10] . Despite reduction of the search space for COMPETING INTERESTS STATEMENTThe authors declare no competing financial interests.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. Supplementary Fig. 1). Synchronized L4-stage wild-type (WT) worms and alg-1(gk214) mutants (hereafter referred to as alg-1(−)), which lack the anti-ALG-1 antibody epitope sequence, were treated with UV irradiation to stabilize in vivo protein-RNA interactions ( Supplementary Fig. 1a). A custom antibody specific for the C. elegans ALG-1 protein ( Supplementary Fig. 1b) was used to enrich for ALG-1 complexes expected to include miRNA and target RNA species. Immunoprecipitated complexes were processed for isolation of sequences protected by ALG-1 protein from nuclease digestion. NIH Public AccessWe obtained 3,864,848 and 5,127,241 reads from WT and alg-1(−) CLIP-seq libraries, respectively, out of which 1,651,523 (42.7%) and 695,895 (13.6%) mapped uniquely to the repeat-masked C. elegans genome ( Supplementary Fig. 2a). Using MIResque, a microRNA ...

SUMMARYMicroRNAs (miRNAs) comprise a large family of small RNA molecules that post-transcriptionally regulate gene expression in many biological pathways1. Most miRNAs are derived from long primary transcripts that undergo processing by Drosha to produce ~65 nucleotide (nt) precursors that are then cleaved by Dicer, resulting in the mature 22 nt forms2,3. Serving as guides in Argonaute protein complexes, mature miRNAs use imperfect base-pairing to recognize sequences in mRNA transcripts, leading to translational repression and destabilization of the target mRNAs4,5. Here we show that the miRNA complex also targets and regulates non-coding RNAs (ncRNAs) that serve as substrates for the miRNA processing pathway. We found that the C. elegans Argonaute, ALG-1, binds to a specific site at the 3′ end of let-7 miRNA primary transcripts and promotes downstream processing events. This interaction is mediated by mature let-7 miRNA via a conserved complementary site in its own primary transcript, thus creating a positive feedback loop. We further show that ALG-1 associates with let-7 primary transcripts in nuclear fractions. Argonaute also binds let-7 primary transcripts in human cells, demonstrating that the miRNA pathway targets non-coding RNAs in addition to protein-coding mRNAs across species. Moreover, our studies in C. elegans reveal a novel role for Argonaute in promoting biogenesis of a targeted transcript, expanding the functions of the miRNA pathway in gene regulation. This discovery of auto-regulation of let-7 biogenesis sets a new paradigm for controlling miRNA expression.

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VOLUME 22 NUMBER 10 OCTOBER 2015 nature structural & molecular biology a r t i c l e s microRNAs (miRs) are a class of small (~22-nt) genomically encoded molecules that inhibit translational initiation and stimulate decay of mRNA targets 1,2 . miRs are transcribed by RNA polymerase II and processed by the RNase III enzymes-Dicer and Drosha with its binding partner, DGCR8-to produce short double-stranded RNAs in the nucleus. One strand associates with the Argonaute (Ago) protein, thus forming the miR-mediated silencing complex (miRISC). miRs guide the pairing of miRISC, with imperfect complementarity, to sequences in target mRNAs, thus resulting in their subsequent destabilization and translational repression of the target 3 . The 'seed sequence' , at nucleotides 2-8, is a key determinant for miRISC-target recognition 4,5 . Recent data have shown that 35-40% of miR-binding sites are found in 3′ untranslated regions (UTRs), 40-50% in coding regions and <5% in 5′-UTR regions of mRNAs 6,7 . More than 60% of the human transcriptome has been predicted to be under miR regulation, thus making this post-transcriptional control pathway as important as protein pathways in the regulation of cell functions 2 . It is clear that miRs have essential roles in regulating diverse functions in normal and diseased cells 8,9 .L1 belongs to the most abundant class of autonomous transposable elements 10 . Human L1 contains two open reading frames, ORF1 and ORF2, which encode a protein with RNA-binding and nucleotide acid-chaperone activity (ORF1) 11 and a protein with endonuclease and reverse-transcriptase activities (ORF2) 12-15 , respectively. L1 mobilizes replicatively from one location in the genome to another by a 'copy-and-paste' mechanism, and it has been proposed to be a remnant of an ancient retrovirus 12,16 . Active and inactive L1s have been implicated in the evolution of mammalian genomes and are linked to cell-based diseases, including cancer [17][18][19] . In addition, somatic L1 insertions are biased toward regions of cancer-specific DNA hypomethylation, thus suggesting that L1 insertions may provide a selective advantage during tumorigenesis 20 . Mechanisms that operate at different levels in gene-expression hierarchies have been selected to control transposition-mediated mutagenesis and mitigate the potential negative effects of newly inserted elements. In germ cells, a specific small-RNA subtype (piwi-interacting RNAs (piRNAs)) efficiently counteracts L1 activity, but these RNAs are not expressed in nongerm cells 21,22 . Somatic cells attenuate element mobilization by DNA methylation of the L1 promoter 23 . Other methods of regulation are mediated by APOBEC proteins 24,25 , microprocessor interactions 26 and Ago-mediated RNA interference in mouse embryonic stem cells 27 . L1-promoter silencing is greatly attenuated, and L1 transcription is reactivated in hypomethylated cells, such as cancer cells and tumor-initiating cells, and is also reactivated during reprogramming [28][29][30] . Because miRs act as regulators of g...