Internal modification of RNAs with N6-methyladenosine (m6A) is a highly conserved means gene expression control. While the METTL3/METTL14 heterodimer adds this mark on thousands transcripts in single-stranded context, substrate requirements and physiological roles second m6A writer METTL16 remain unknown. Here we describe crystal structure human to reveal methyltransferase domain furnished an extra N-terminal module, which together form deep-cut groove that essential for RNA binding. When...

inhibition by 3 0 splice site m 6 A is conserved in mammals

PIWI proteins and PIWI-interacting RNAs (piRNAs) mediate repression of transposons in the animal gonads. Primary processing converts long single-stranded into ∼30-nt piRNAs, but their entry biogenesis pathway is unknown. Here, we demonstrate that an RNA element at 5′ end a piRNA cluster—which termed trigger sequence (PTS)—can induce primary any downstream sequence. We propose such signals are triggers for generation original pool piRNAs. also endonucleolytic cleavage transcript by cytosolic...

The functional consequence of N6-methyladenosine (m6A) RNA modification is mediated by "reader" proteins the YTH family. domain-containing 2 (YTHDC2) essential for mammalian fertility, but its molecular function poorly understood. Here, we identify U-rich motifs as binding sites YTHDC2 on 3′ UTRs mouse testicular targets. Although domain an m6A-binder in vitro, point mutant mice are fertile. Significantly, loss 3′→5′ helicase activity causes infertility, with catalytic-dead mutation being...

The Kcnq1 imprinting control region (ICR) located in intron 10 of the gene is unmethylated on paternal chromosome and methylated maternal has been implicated manifestation parent-of-origin-specific expression six neighboring genes. ICR harbors bidirectional silencer activity drives an antisense RNA, Kcnq1ot1, which overlaps coding region. To elucidate whether Kcnq1ot1 RNA plays a role silencing ICR, we have characterized factor binding sites by genomic footprinting tested functional...

The Kcnq1ot1 antisense noncoding RNA has been implicated in long-range bidirectional silencing, but the underlying mechanisms remain enigmatic.Here we characterize a domain at 5 end of that carries out transcriptional silencing linked genes using an episomal vector system.The property maps to highly conserved repeat motif within domain, which directs by interaction with chromatin, resulting histone H3 lysine 9 trimethylation.Intriguingly, is also required target perinucleolar compartment...

Significance Large parts of eukaryotic genomes are composed transposons. Mammalian use DNA methylation to silence these genomic parasites. A class small RNAs called Piwi-interacting (piRNAs) is used specifically guide the machinery transposon elements. How germ cells make piRNAs not entirely understood. We identify a mouse protein and demonstrate its importance for silencing. find that collaborates with other factors already implicated in piRNA production. Moreover, required production...

Piwi-interacting RNAs (piRNAs) guide Piwi Argonautes to suppress transposon activity in animal gonads. Known piRNA populations are extremely complex, with millions of individual sequences present a single organism. Despite this complexity, specific proteins incorporate piRNAs distinct nucleotide- and strand-biases (antisense or sense) unknown origin. Here, we examined the contribution structural domains toward defining these biases. We report first crystal structure MID domain from Argonaute...

Small RNAs called PIWI-interacting (piRNAs) act as an immune system to suppress transposable elements in the animal gonads. A poorly understood adaptive pathway links cytoplasmic slicing of target RNA by PIWI protein MILI loading target-derived piRNAs into nuclear MIWI2. Here we demonstrate that generates a 16-nt by-product is discarded and pre-piRNA intermediate used for phased piRNA production. The ATPase activity Mouse Vasa Homolog (MVH) essential processing piRNAs, ensuring transposon...

PIWI-interacting RNAs (piRNAs) guide PIWI proteins to suppress transposons in the cytoplasm and nucleus of animal germ cells, but how silencing two compartments is coordinated not known. Here we demonstrate that endonucleolytic slicing a transcript by cytosolic mouse protein MILI acts as trigger initiate its further 5'→3' processing into non-overlapping fragments. These fragments accumulate new piRNAs within both nuclear MIWI2. We also identify Exonuclease domain-containing 1 (EXD1) partner...

The 5' end of eukaryotic mRNAs is protected by the m7G-cap structure. transcription start site nucleotide ribose methylated (Nm) in many eukaryotes, whereas an adenosine at this position further N6 (m6A) mammalian Phosphorylated C-terminal domain (CTD)-interacting Factor 1 (PCIF1) to generate m6Am. Here, we show that although loss cap-specific m6Am mice does not affect viability or fertility, Pcif1 mutants display reduced body weight. Transcriptome analyses mutant mouse tissues support a...

Small RNAs called PIWI -interacting (piRNAs) are essential for transposon control and fertility in animals. Primary processing is the small RNA biogenesis pathway that uses long single-stranded precursors to generate millions of individual piRNAs, but molecular mechanisms identify a transcript as precursor poorly understood. Here we demonstrate artificial tethering piRNA factor, Armi, sufficient direct it into primary Drosophila ovaries an ovarian cell culture model. In fly somatic follicle...

DNA methylation is a major silencing mechanism of transposable elements (TEs). Here we report that TEX15, testis-specific protein, required for TE silencing. TEX15 expressed in embryonic germ cells and functions during genome-wide epigenetic reprogramming. The Tex15 mutant exhibits hypomethylation TEs at level similar to Mili Dnmt3c but not Miwi2 mutants. associated with MILI testis. As loss causes desilencing intact piRNA production, our results identify as new essential regulator may...

Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs. piRNAs protect the genome integrity germline by silencing active transposable elements and essential for germ cell development. Most piRNA pathway proteins evolutionarily conserved. MOV10L1, testis-specific RNA helicase, binds to precursors is master regulator biogenesis in mouse. Here we report that mutation MOV10L1 ATP hydrolysis site leads depletion on Piwi proteins, de-repression elements, conglomeration into polar...

Piwi-interacting RNAs (piRNAs) guide Piwi argonautes to their transposon targets for silencing. The highly conserved protein Maelstrom is linked both piRNA biogenesis and effector roles in this pathway. One defining feature of the predicted MAEL domain unknown molecular function. Here, we present first crystal structure from Bombyx Maelstrom, which reveals a nuclease fold. overall architecture resembles that found Mg 2+ - or Mn -dependent DEDD nucleases, but clear distinguishing presence...

PIWI proteins and their associated small RNAs, called PIWI-interacting RNAs (piRNAs), restrict transposon activity in animal gonads to ensure fertility. Distinct biogenesis pathways load piRNAs into the MILI MIWI2 mouse male embryonic germline. While most are derived via a slicer-independent pathway, slicing loads with series of phased piRNAs. Tudor domain-containing 12 (TDRD12) its interaction partner Exonuclease 1 (EXD1) required for loading MIWI2, but only Tdrd12 is essential fertility,...

Eukaryotic mRNAs are modified at the 5′ end with a methylated guanosine (m 7 G) that is attached to transcription start site (TSS) nucleotide. The TSS nucleotide 2′- O -methylated (Nm) by CMTR1 in organisms ranging from insects human. In mammals, adenosine can be further N 6 RNA polymerase II phosphorylated CTD-interacting factor 1 (PCIF1) create m Am. Curiously, fly ortholog of mammalian PCIF1 demonstrated catalytic-dead, and its functions not known. Here, we show Pcif1 mutant flies display...

Antisense transcription has been shown to be one of the hierarchies that control gene expression in eukaryotes. Recently, we have documented mouse Kcnq1 imprinting region (ICR) harbors bidirectional silencing property, and this feature is linked an antisense RNA, Kcnq1ot1. In investigation, using genomic footprinting, identified three NF-Y factor binding sites appearing a methylation-sensitive manner Kcnq1ot1 promoter. By employing dominant negative mutant factor, positively regulates...