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The Mammalian Cap-Specific m6Am RNA Methyltransferase PCIF1 Regulates Transcript Levels in Mouse Tissues.
Cell Rep, 2020 Aug 18; 32 (7): 108038
The 5' end of eukaryotic mRNAs is protected by the mG-cap structure. The transcription start site nucleotide is ribose methylated (Nm) in many eukaryotes, whereas an adenosine at this position is further methylated at the N position (mA) by the mammalian Phosphorylated C-terminal domain (CTD)-interacting Factor 1 (PCIF1) to generate mAm. Here, we show that although the loss of cap-specific mAm in mice does not affect viability or fertility, the Pcif1 mutants display reduced body weight. Transcriptome analyses of mutant mouse tissues support a role for the cap-specific mAm modification in stabilizing transcripts. In contrast, the Drosophila Pcif1 is catalytically dead, but like its mammalian counterpart, it retains the ability to associate with the Ser5-phosphorylated CTD of RNA polymerase II (RNA Pol II). Finally, we show that the Trypanosoma Pcif1 is an mAm methylase that contributes to the N,N,2'-O-trimethyladenosine (mAm) in the hypermethylated cap4 structure of trypanosomatids. Thus, PCIF1 has evolved to function in catalytic and non-catalytic roles.
TEX15 associates with MILI and silences transposable elements in male germ cells.
Genes Dev, 2020 Jun 1; 34 (11-12): 745-750
DNA methylation is a major silencing mechanism of transposable elements (TEs). Here we report that TEX15, a testis-specific protein, is required for TE silencing. TEX15 is expressed in embryonic germ cells and functions during genome-wide epigenetic reprogramming. The mutant exhibits DNA hypomethylation in TEs at a level similar to and but not mutants. TEX15 is associated with MILI in testis. As loss of causes TE desilencing with intact piRNA production, our results identify TEX15 as a new essential epigenetic regulator that may function as a nuclear effector of MILI to silence TEs by DNA methylation.
Decapping Enzyme NUDT12 Partners with BLMH for Cytoplasmic Surveillance of NAD-Capped RNAs.
Cell Rep, 2019 Dec 24; 29 (13): 4422-4434.e13
RNA polymerase II transcripts receive a protective 5',5'-triphosphate-linked 7-methylguanosine (mG) cap, and its removal by decapping enzymes like DCP2 is critical for initiation of RNA decay. Alternative RNA caps can be acquired when transcription initiation uses metabolites like nicotinamide adenine dinucleotide (NAD), generating NAD-RNAs. Here, we identify human NUDT12 as a cytosolic NAD-RNA decapping enzyme. NUDT12 is active only as homodimers, with each monomer contributing to creation of the two functional catalytic pockets. We identify an ∼600-kDa dodecamer complex between bleomycin hydrolase (BLMH) and NUDT12, with BLMH being required for localization of NUDT12 to a few discrete cytoplasmic granules that are distinct from P-bodies. Both proteins downregulate gene expression when artificially tethered to a reporter RNA in vivo. Furthermore, loss of Nudt12 results in a significant upregulation of circadian clock transcripts in mouse liver. Overall, our study points to a physiological role for NUDT12 in the cytosolic surveillance of NAD-RNAs.
Nxf3: a middleman with the right connections for unspliced piRNA precursor export.
Genes Dev, 2019 Sep 1; 33 (17-18): 1095-1097
RNA export is tightly coupled to splicing in metazoans. In the germline, precursors for the majority of Piwi-interacting RNAs (piRNAs) are unspliced. In this issue of , Kneuss and colleagues (pp. 1208-1220) identify Nxf3 as a novel germline-specific export adapter for such unspliced transcripts. Their findings reveal the sequence of events leading from its role at the site of transcription to delivery of the cargo to cytoplasmic piRNA biogenesis sites.
An RNA exporter that enforces a no-export policy.
Nat Struct Mol Biol, 2019 Sep; 26 (9): 758-759
Counting the Cuts: MAZTER-Seq Quantifies m6A Levels Using a Methylation-Sensitive Ribonuclease.
Cell, 2019 Jul 25; 178 (3): 515-517
Garcia-Campos et al. describe MAZTER-seq, which deploys a sequence-specific, methylation-sensitive bacterial single-stranded ribonuclease MazF to provide nucleotide-resolution quantification of mA methylation sites. The study reveals many new sites and supports the idea of a predictable "mA code," where methylation levels are dictated primarily by local sequence at the site of methylation.
Methylation of Structured RNA by the m6A Writer METTL16 Is Essential for Mouse Embryonic Development.
Mol Cell, 2018 Sep 20; 71 (6): 986-1000.e11
Internal modification of RNAs with N-methyladenosine (mA) is a highly conserved means of gene expression control. While the METTL3/METTL14 heterodimer adds this mark on thousands of transcripts in a single-stranded context, the substrate requirements and physiological roles of the second mA writer METTL16 remain unknown. Here we describe the crystal structure of human METTL16 to reveal a methyltransferase domain furnished with an extra N-terminal module, which together form a deep-cut groove that is essential for RNA binding. When presented with a random pool of RNAs, METTL16 selects for methylation-structured RNAs where the critical adenosine is present in a bulge. Mouse 16-cell embryos lacking Mettl16 display reduced mRNA levels of its methylation target, the SAM synthetase Mat2a. The consequence is massive transcriptome dysregulation in ∼64-cell blastocysts that are unfit for further development. This highlights the role of an mA RNA methyltransferase in facilitating early development via regulation of SAM availability.
Exonuclease Domain-Containing 1 Enhances MIWI2 piRNA Biogenesis via Its Interaction with TDRD12.
Cell Rep, 2018 Sep 25; 24 (13): 3423-3432.e4
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 PIWI proteins MILI and MIWI2 in the mouse male embryonic germline. While most MILI piRNAs are derived via a slicer-independent pathway, MILI slicing loads MIWI2 with a series of phased piRNAs. Tudor domain-containing 12 (TDRD12) and its interaction partner Exonuclease domain-containing 1 (EXD1) are required for loading MIWI2, but only Tdrd12 is essential for fertility, leaving us with no explanation for the physiological role of Exd1. Using an artificial piRNA precursor, we demonstrate that MILI-triggered piRNA biogenesis is greatly reduced in the Exd1 mutant. The situation deteriorates in the sensitized Exd1 mutant (Exd1;Tdrd12), where diminished MIWI2 piRNA levels de-repress LINE1 retrotransposons, leading to infertility. Thus, EXD1 enhances MIWI2 piRNA biogenesis via a functional interaction with TDRD12.
Transposon silencing in the Drosophila female germline is essential for genome stability in progeny embryos.
Life Sci Alliance, 2018 Oct; 1 (5): e201800179
The Piwi-interacting RNA pathway functions in transposon control in the germline of metazoans. The conserved RNA helicase Vasa is an essential Piwi-interacting RNA pathway component, but has additional important developmental functions. Here, we address the importance of Vasa-dependent transposon control in the female germline and early embryos. We find that transient loss of expression during early oogenesis leads to transposon up-regulation in supporting nurse cells of the fly egg-chamber. We show that elevated transposon levels have dramatic consequences, as de-repressed transposons accumulate in the oocyte where they cause DNA damage. We find that suppression of Chk2-mediated DNA damage signaling in mutant females restores oogenesis and egg production. Damaged DNA and up-regulated transposons are transmitted from the mother to the embryos, which sustain severe nuclear defects and arrest development. Our findings reveal that the Vasa-dependent protection against selfish genetic elements in the nuage of nurse cell is essential to prevent DNA damage-induced arrest of embryonic development.
Regulation of m6A Transcripts by the 3'→5' RNA Helicase YTHDC2 Is Essential for a Successful Meiotic Program in the Mammalian Germline.
Mol Cell, 2017 Oct 19; 68 (2): 374-387.e12
N-methyladenosine (mA) is an essential internal RNA modification that is critical for gene expression control in most organisms. Proteins with a YTH domain recognize mA marks and are mediators of molecular functions like RNA splicing, mRNA decay, and translation control. Here we demonstrate that YTH domain-containing 2 (YTHDC2) is an mA reader that is essential for male and female fertility in mice. High-throughput mapping of the mA transcriptome and expression analysis in the Yhtdc2 mutant testes reveal an upregulation of mA-enriched transcripts. Our biochemical studies indicate that YTHDC2 is an RNA-induced ATPase with a 3'→5' RNA helicase activity. Furthermore, YTHDC2 recruits the 5'→3' exoribonuclease XRN1 via Ankyrin repeats that are inserted in between the RecA modules of the RNA helicase domain. Our studies reveal a role for YTHDC2 in modulating the levels of mA-modified germline transcripts to maintain a gene expression program that is conducive for progression through meiosis.
Selective termination of lncRNA transcription promotes heterochromatin silencing and cell differentiation.
EMBO J, 2017 Sep 1; 36 (17): 2626-2641
Long non-coding RNAs (lncRNAs) regulating gene expression at the chromatin level are widespread among eukaryotes. However, their functions and the mechanisms by which they act are not fully understood. Here, we identify new fission yeast regulatory lncRNAs that are targeted, at their site of transcription, by the YTH domain of the RNA-binding protein Mmi1 and degraded by the nuclear exosome. We uncover that one of them, nam1, regulates entry into sexual differentiation. Importantly, we demonstrate that Mmi1 binding to this lncRNA not only triggers its degradation but also mediates its transcription termination, thus preventing lncRNA transcription from invading and repressing the downstream gene encoding a mitogen-activated protein kinase kinase kinase (MAPKKK) essential to sexual differentiation. In addition, we show that Mmi1-mediated termination of lncRNA transcription also takes place at pericentromeric regions where it contributes to heterochromatin gene silencing together with RNA interference (RNAi). These findings reveal an important role for selective termination of lncRNA transcription in both euchromatic and heterochromatic lncRNA-based gene silencing processes.
Recruitment of Armitage and Yb to a transcript triggers its phased processing into primary piRNAs in Drosophila ovaries.
PLoS Genet, 2017 Aug; 13 (8): e1006956
Small RNAs called PIWI -interacting RNAs (piRNAs) are essential for transposon control and fertility in animals. Primary processing is the small RNA biogenesis pathway that uses long single-stranded RNA precursors to generate millions of individual piRNAs, but the molecular mechanisms that identify a transcript as a precursor are poorly understood. Here we demonstrate that artificial tethering of the piRNA biogenesis factor, Armi, to a transcript is sufficient to direct it into primary processing in Drosophila ovaries and in an ovarian cell culture model. In the fly ovarian somatic follicle cells, the transcript becomes cleaved in a stepwise manner, with a 5'→3' directionality, liberating U1-containing ~24 nt piRNAs that are loaded into Piwi. Although uridines are preferred for generation of piRNA 5' ends, processing takes place even in their absence, albeit at a lower efficiency. We show that recombinant Armi has 5'→3' helicase activity, and mutations that abolish this activity also reduce piRNA processing in vivo. Another somatic piRNA pathway factor Yb, an interactor of Armi, is also able to trigger piRNA biogenesis when tethered to a transcript. Tethering-mediated primary piRNA biogenesis is also functional in the fly ovarian germline and loads all the three PIWI proteins present in this environment. Our study finds a broad correlation between piRNA processing and localization of the tethered factors to the cytoplasmic perinuclear ribonucleoprotein granules called germline nuage or somatic Yb bodies. We conclude that transcripts bound by Armi and Yb are identified as piRNA precursors, resulting in localization to cytoplasmic processing granules and their subsequent engagement by the resident piRNA biogenesis machinery.
Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function.
Dev Cell, 2017 Jun 19; 41 (6): 623-637.e9
Small RNAs called PIWI-interacting RNAs (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 the PIWI protein MILI to loading of target-derived piRNAs into nuclear MIWI2. Here we demonstrate that MILI slicing generates a 16-nt by-product that is discarded and a pre-piRNA intermediate that is used for phased piRNA production. The ATPase activity of Mouse Vasa Homolog (MVH) is essential for processing the intermediate into piRNAs, ensuring transposon silencing and male fertility. The ATPase activity controls dissociation of an MVH complex containing PIWI proteins, piRNAs, and slicer products, allowing safe handover of the intermediate. In contrast, ATPase activity of TDRD9 is dispensable for piRNA biogenesis but is essential for transposon silencing and male fertility. Our work implicates distinct RNA helicases in specific steps along the nuclear piRNA pathway.
Characterization of the mammalian RNA exonuclease 5/NEF-sp as a testis-specific nuclear 3' → 5' exoribonuclease.
RNA, 2017 Sep; 23 (9): 1385-1392
Ribonucleases catalyze maturation of functional RNAs or mediate degradation of cellular transcripts, activities that are critical for gene expression control. Here we identify a previously uncharacterized mammalian nuclease family member NEF-sp (RNA exonuclease 5 [REXO5] or LOC81691) as a testis-specific factor. Recombinant human NEF-sp demonstrates a divalent metal ion-dependent 3' → 5' exoribonuclease activity. This activity is specific to single-stranded RNA substrates and is independent of their length. The presence of a 2'-O-methyl modification at the 3' end of the RNA substrate is inhibitory. Ectopically expressed NEF-sp localizes to the nucleolar/nuclear compartment in mammalian cell cultures and this is dependent on an amino-terminal nuclear localization signal. Finally, mice lacking NEF-sp are viable and display normal fertility, likely indicating overlapping functions with other nucleases. Taken together, our study provides the first biochemical and genetic exploration of the role of the NEF-sp exoribonuclease in the mammalian genome.
Mutations in the MOV10L1 ATP Hydrolysis Motif Cause piRNA Biogenesis Failure and Male Sterility in Mice.
Biol Reprod, 2016 Nov 1; 95 (5): 103
Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs. piRNAs protect the genome integrity of the germline by silencing active transposable elements and are essential for germ cell development. Most piRNA pathway proteins are evolutionarily conserved. MOV10L1, a testis-specific RNA helicase, binds to piRNA precursors and is a master regulator of piRNA biogenesis in mouse. Here we report that mutation of the MOV10L1 ATP hydrolysis site leads to depletion of piRNAs on Piwi proteins, de-repression of transposable elements, and conglomeration of piRNA pathway proteins into polar granules. The Mov10l1 mutant mice exhibit meiotic arrest and male sterility. Our results show that mutation of the MOV10L1 ATP hydrolysis site perturbs piRNA biogenesis.
PIWI Slicing and EXD1 Drive Biogenesis of Nuclear piRNAs from Cytosolic Targets of the Mouse piRNA Pathway.
Mol Cell, 2016 Jan 7; 61 (1): 138-152
PIWI-interacting RNAs (piRNAs) guide PIWI proteins to suppress transposons in the cytoplasm and nucleus of animal germ cells, but how silencing in the two compartments is coordinated is not known. Here we demonstrate that endonucleolytic slicing of a transcript by the cytosolic mouse PIWI protein MILI acts as a trigger to initiate its further 5'→3' processing into non-overlapping fragments. These fragments accumulate as new piRNAs within both cytosolic MILI and the nuclear MIWI2. We also identify Exonuclease domain-containing 1 (EXD1) as a partner of the MIWI2 piRNA biogenesis factor TDRD12. EXD1 homodimers are inactive as a nuclease but function as an RNA adaptor within a PET (PIWI-EXD1-Tdrd12) complex. Loss of Exd1 reduces sequences generated by MILI slicing, impacts biogenesis of MIWI2 piRNAs, and de-represses LINE1 retrotransposons. Thus, piRNA biogenesis triggered by PIWI slicing, and promoted by EXD1, ensures that the same guides instruct PIWI proteins in the nucleus and cytoplasm.
PIWI Slicing and RNA Elements in Precursors Instruct Directional Primary piRNA Biogenesis.
Cell Rep, 2015 Jul 21; 12 (3): 418-428
PIWI proteins and PIWI-interacting RNAs (piRNAs) mediate repression of transposons in the animal gonads. Primary processing converts long single-stranded RNAs into ∼30-nt piRNAs, but their entry into the biogenesis pathway is unknown. Here, we demonstrate that an RNA element at the 5' end of a piRNA cluster—which we termed piRNA trigger sequence (PTS)—can induce primary processing of any downstream sequence. We propose that such signals are triggers for the generation of the original pool of piRNAs. We also demonstrate that endonucleolytic cleavage of a transcript by a cytosolic PIWI results in its entry into primary processing, which triggers the generation of non-overlapping, contiguous primary piRNAs in the 3' direction from the target transcript. These piRNAs are loaded into a nuclear PIWI, thereby linking cytoplasmic post-transcriptional silencing to nuclear transcriptional repression.
Metazoan Maelstrom is an RNA-binding protein that has evolved from an ancient nuclease active in protists.
RNA, 2015 May; 21 (5): 833-839
Piwi-interacting RNAs (piRNAs) guide Piwi argonautes to their transposon targets for silencing. The highly conserved protein Maelstrom is linked to both piRNA biogenesis and effector roles in this pathway. One defining feature of Maelstrom is the predicted MAEL domain of unknown molecular function. Here, we present the first crystal structure of the MAEL domain from Bombyx Maelstrom, which reveals a nuclease fold. The overall architecture resembles that found in Mg(2+)- or Mn(2+)-dependent DEDD nucleases, but a clear distinguishing feature is the presence of a structural Zn(2+) ion coordinated by the conserved ECHC residues. Strikingly, metazoan Maelstrom orthologs across the animal kingdom lack the catalytic DEDD residues, and as we show for Bombyx Maelstrom are inactive as nucleases. However, a MAEL domain-containing protein from amoeba having both sequence motifs (DEDD and ECHC) is robustly active as an exoribonuclease. Finally, we show that the MAEL domain of Bombyx Maelstrom displays a strong affinity for single-stranded RNAs. Our studies suggest that the ancient MAEL nuclease domain evolved to function as an RNA-binding module in metazoan Maelstrom.
The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing.
Genes Dev, 2015 Mar 15; 29 (6): 617-629
Piwi-piRNA (Piwi-interacting RNA) ribonucleoproteins (piRNPs) enforce retrotransposon silencing, a function critical for preserving the genome integrity of germ cells. The molecular functions of most of the factors that have been genetically implicated in primary piRNA biogenesis are still elusive. Here we show that MOV10L1 exhibits 5'-to-3' directional RNA-unwinding activity in vitro and that a point mutation that abolishes this activity causes a failure in primary piRNA biogenesis in vivo. We demonstrate that MOV10L1 selectively binds piRNA precursor transcripts and is essential for the generation of intermediate piRNA processing fragments that are subsequently loaded to Piwi proteins. Multiple analyses suggest an intimate coupling of piRNA precursor processing with elements of local secondary structures such as G quadruplexes. Our results support a model in which MOV10L1 RNA helicase activity promotes unwinding and funneling of the single-stranded piRNA precursor transcripts to the endonuclease that catalyzes the first cleavage step of piRNA processing.
Fly piRNA biogenesis: tap dancing with Tej.
BMC Biol, 2014 Oct 6; 12 : 77
Piwi-interacting RNAs (piRNAs) protect animal germlines from the deleterious effects of transposon activity. Unlike other small RNA classes like microRNAs (miRNAs) and small interfering RNAs (siRNAs), an exceptionally large number of factors are implicated in the biogenesis of piRNAs. Kai et al. have now added another one to this growing list, which we discuss in the overall context of our current knowledge of the piRNA biogenesis pathway in the Drosophila ovarian germline. See research article: http://www.biomedcentral.com/1741-7007/12/61.