SUMMARY The shoot apical meristem (SAM) comprises a group of undifferentiated cells that divide to maintain the meristem and also give rise to all plant shoot organs. SAM fate is specified by HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP) transcription factors, which are targets of miR166/165. In Arabidopsis, AGO10 is a critical regulator of SAM maintenance, but the mechanism of regulation remains unknown. Here we demonstrate that AGO10 specifically recruits miR166/165. The AGO10-miR166/165 association is determined by the distinct structure of the miR166/165 duplex. Deficient loading of miR166 into AGO10 results in a defective SAM. AGO10 has a higher binding affinity for miR166 than does AGO1, a master repressor for miRNA targets. Notably, the miR166/165-binding ability of AGO10, but not its catalytic activity, is required for SAM development. We propose that AGO10 functions as a decoy for miR166/165 to maintain the SAM, preventing their incorporation into AGO1 complexes and the subsequent repression of HD-ZIP gene expression.

SUMMARY RIG-I is a cytosolic sensor of viral RNA that plays crucial roles in the induction of type I interferons. The C-terminal domain (CTD) of RIG-I is responsible for the recognition of viral RNA with 5′ triphosphate (5′ ppp). However, the mechanism of viral RNA recognition by RIG-I is still not fully understood. Here we show that RIG-I CTD binds 5′ ppp dsRNA or ssRNA, as well as blunt-ended dsRNA, and exhibits the highest affinity for 5′ ppp dsRNA. Crystal structures of RIG-I CTD bound to 5′ ppp dsRNA with GC- and AU- rich sequences revealed that RIG-I recognizes the termini of the dsRNA and interacts with the 5′ triphosphate through extensive electrostatic interactions. Mutagenesis and RNA binding studies demonstrated that similar binding surfaces are involved in the recognition of different forms of RNA. Mutations of key residues at the RNA binding surface affected RIG-I signaling in cells.

SUMMARY RNA polymerase II (RNAPII) lies at the core of dynamic control of gene expression. Using 53 RNAPII point mutants, we generated a point mutant epistatic miniarray profile (pE-MAP) comprising ~60,000 quantitative genetic interactions in Saccharomyces cerevisiae. This analysis enabled functional assignment of RNAPII subdomains and uncovered connections between individual regions and other protein complexes. Using splicing microarrays and mutants that alter elongation rates in vitro, we found an inverse relationship between RNAPII speed and in vivo splicing efficiency. Furthermore, the pE-MAP classified fast and slow mutants that favor upstream and downstream start site selection, respectively. The striking coordination of polymerization rate with transcription initiation and splicing suggests that transcription rate is tuned to regulate multiple gene expression steps. The pE-MAP approach provides a powerful strategy to understand other multifunctional machines at amino acid resolution.

Intragenomic conflicts are fueled by rapidly evolving selfish genetic elements, which induce selective pressures to innovate opposing repressive mechanisms. This is patently manifest in sex-ratio (SR) meiotic drive systems, in which distorter and suppressor factors bias and restore equal transmission of X and Y sperm. Here, we reveal that multiple SR suppressors in Drosophila simulans (Nmy and Tmy) encode related hairpin RNAs (hpRNAs), which generate endo-siRNAs that repress the paralogous distorters Dox and MDox. All components in this drive network are recently evolved and largely testis restricted. To connect SR hpRNA function to the RNAi pathway, we generated D. simulans null mutants of Dcr-2 and AGO2. Strikingly, these core RNAi knockouts massively derepress Dox and MDox and are in fact completely male sterile and exhibit highly defective spermatogenesis. Altogether, our data reveal how the adaptive capacity of hpRNAs is critically deployed to restrict selfish gonadal genetic systems that can exterminate a species.

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.

RNA-induced silencing complex (RISC) is composed of miRNAs and AGO proteins. AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5' and 3' RNA fragments. The 5' cleaved RNA fragments are marked with uridylation for degradation. Here, we identified novel cofactors of Arabidopsis AGOs, named RICE1 and RICE2. RICE proteins specifically degraded single-strand (ss) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo. RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits. Notably, ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5' cleavage RISC fragments with extended uridine tails. We conclude that RICEs act to degrade uridylated 5’ products of AGO cleavage to maintain functional RISC. Our study also suggests a possible link between decay of cleaved target mRNAs and miRNA stability in RISC.DOI: http://dx.doi.org/10.7554/eLife.24466.001

Post-transcriptional regulatory strategies that involve coupling between terminal uridyltransferase (TUTase) and exoribonuclease enzymes have recently been characterized in diverse species. Of note, the 3' exoribonuclease Dis3L2 has received substantial attention as a factor that metabolizes uridylated substrates in contexts such as general mRNA degradation, turnover of specific miRNAs, and quality control of noncoding RNAs. To date, most studies of Dis3L2 have focused on fungi and mammalian cells. Here, we introduce as a system that permits analysis of molecular mechanisms as well as the ability to interrogate organismal phenotypes. We started with a structure-function analysis of the TUTase Tailor, which we recently identified to inhibit biogenesis of splicing-derived miRNA hairpins. Next, we show that Tailor/Dis3L2 form a complex via N-terminal domains in the respective proteins that are distinct from their catalytic domains. In vitro, Dis3L2 has nuclease activity, but substrate oligouridylation by Tailor stimulates their degradation by Dis3L2, especially for structured substrates. We analyzed mutants of and, which are viable and lack overt morphological defects. Instead, these mutants exhibit defects in female and male fertility, implying specific requirements in the germline. defects are more severe than, and their requirements appear stronger in males than in females. In particular, loss of Dis3L2 completely blocks productive spermatogenesis, causing male sterility. RNA-seq analysis from single- and double-mutant testes reveals aberrant gene expression programs and suggests that noncoding RNAs may be preferentially affected by Dis3L2. Overall, our studies of a new tailing/trimming complex reveal unexpectedly specific requirements during gametogenesis.

Photoreceptors in the crystalline eye are recruited by receptor tyrosine kinase (RTK)/Ras signaling mediated by Epidermal growth factor receptor (EGFR) and the Sevenless (Sev) receptor. Analyses of an allelic deletion series of the locus, along with a panel of modified genomic rescue transgenes, show that eye patterning depends on both miRNAs. Transcriptional reporter and activity sensor transgenes reveal expression and function of miR-279/996 in non-neural cells of the developing eye. Moreover, mutants exhibit substantial numbers of ectopic photoreceptors, particularly of R7, and cone cell loss. These miRNAs restrict RTK signaling in the eye, since nulls are dominantly suppressed by positive components of the EGFR pathway and enhanced by heterozygosity for an EGFR repressor. miR-279/996 limit photoreceptor recruitment by targeting multiple positive RTK/Ras signaling components that promote photoreceptor/R7 specification. Strikingly, deletion of sufficiently derepresses RTK/Ras signaling so as to rescue a population of R7 cells in R7-specific RTK null mutants and, which otherwise completely lack this cell fate. Altogether, we reveal a rare setting of developmental cell specification that involves substantial miRNA control.