Splice site m6A methylation prevents binding of U2AF35 to inhibit RNA splicing.

The N6-methyladenosine (m6A) RNA modification is used widely to alter the fate of mRNAs. Here we demonstrate that the C. elegans writer METT-10 (the ortholog of mouse METTL16) deposits an m6A mark on the 3' splice site (AG) of the S-adenosylmethionine (SAM) synthetase pre-mRNA, which inhibits its proper splicing and protein production. The mechanism is triggered by a rich diet and acts as an m6A-mediated switch to stop SAM production and regulate its homeostasis. Although the mammalian SAM synthetase pre-mRNA is not regulated via this mechanism, we show that splicing inhibition by 3' splice site m6A is conserved in mammals. The modification functions by physically preventing the essential splicing factor U2AF35 from recognizing the 3' splice site. We propose that use of splice-site m6A is an ancient mechanism for splicing regulation.

Counting the Cuts: MAZTER-Seq Quantifies m6A Levels Using a Methylation-Sensitive Ribonuclease.

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 m6A methylation sites. The study reveals many new sites and supports the idea of a predictable "m6A code," where methylation levels are dictated primarily by local sequence at the site of methylation.

The XRN1-regulated RNA helicase activity of YTHDC2 ensures mouse fertility independently of m6A recognition.

The functional consequence of N6-methyladenosine (m6A) RNA modification is mediated by "reader" proteins of the YTH family. YTH domain-containing 2 (YTHDC2) is essential for mammalian fertility, but its molecular function is poorly understood. Here, we identify U-rich motifs as binding sites of YTHDC2 on 3' UTRs of mouse testicular RNA targets. Although its YTH domain is an m6A-binder in vitro, the YTH point mutant mice are fertile. Significantly, the loss of its 3'→5' RNA helicase activity causes mouse infertility, with the catalytic-dead mutation being dominant negative. Biochemical studies reveal that the weak helicase activity of YTHDC2 is enhanced by its interaction with the 5'→3' exoribonuclease XRN1. Single-cell transcriptomics indicate that Ythdc2 mutant mitotic germ cells transition into meiosis but accumulate a transcriptome with mixed mitotic/meiotic identity that fail to progress further into meiosis. Finally, our demonstration that ythdc2 mutant zebrafish are infertile highlights its conserved role in animal germ cell development.

TEX15 associates with MILI and silences transposable elements in male germ cells.

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 Tex15 mutant exhibits DNA hypomethylation in TEs at a level similar to Mili and Dnmt3c but not Miwi2 mutants. TEX15 is associated with MILI in testis. As loss of Tex15 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.

Methylation of Structured RNA by the m6A Writer METTL16 Is Essential for Mouse Embryonic Development.

Internal modification of RNAs with N6-methyladenosine (m6A) 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 m6A 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 m6A RNA methyltransferase in facilitating early development via regulation of SAM availability.

In vivo PIWI slicing in mouse testes deviates from rules established in vitro.

Argonautes are small RNA-binding proteins, with some having small RNA-guided endonuclease (slicer) activity that cleaves target nucleic acids. One cardinal rule that is structurally defined is the inability of slicers to cleave target RNAs when nucleotide mismatches exist between the paired small RNA and the target at the cleavage site. Animal-specific PIWI clade Argonautes associate with PIWI-interacting RNAs (piRNAs) to silence transposable elements in the gonads, and this is essential for fertility. We previously demonstrated that purified endogenous mouse MIWI fails to cleave mismatched targets in vitro. Surprisingly, here we find using knock-in mouse models that target sites with cleavage-site mismatches at the 10th and 11th piRNA nucleotides are precisely sliced in vivo. This is identical to the slicing outcome in knock-in mice where targets are base-paired perfectly with the piRNA. Additionally, we find that pachytene piRNA-guided slicing in both these situations failed to initiate phased piRNA production from the specific target mRNA we studied. Instead, the two slicer cleavage fragments were retained in PIWI proteins as pre-piRNA and 17-19 nt by-product fragments. Our results indicate that PIWI slicing rules established in vitro are not respected in vivo, and that all targets of PIWI slicing are not substrates for piRNA biogenesis.

The catalytic-dead Pcif1 regulates gene expression and fertility in Drosophila.

Eukaryotic mRNAs are modified at the 5' end with a methylated guanosine (m7G) that is attached to the transcription start site (TSS) nucleotide. The TSS nucleotide is 2'-O-methylated (Nm) by CMTR1 in organisms ranging from insects to human. In mammals, the TSS adenosine can be further N 6 -methylated by RNA polymerase II phosphorylated CTD-interacting factor 1 (PCIF1) to create m6Am. Curiously, the fly ortholog of mammalian PCIF1 is demonstrated to be catalytic-dead, and its functions are not known. Here, we show that Pcif1 mutant flies display a reduced fertility which is particularly marked in females. Deep sequencing analysis of Pcif1 mutant ovaries revealed transcriptome changes with a notable increase in expression of genes belonging to the mitochondrial ATP synthetase complex. Furthermore, the Pcif1 protein is distributed along euchromatic regions of polytene chromosomes, and the Pcif1 mutation behaved as a modifier of position-effect-variegation (PEV) suppressing the heterochromatin-dependent silencing of the white gene. Similar or stronger changes in the transcriptome and PEV phenotype were observed in flies that expressed a cytosolic version of Pcif1. These results point to a nuclear cotranscriptional gene regulatory role for the catalytic-dead fly Pcif1 that is probably based on its conserved ability to interact with the RNA polymerase II carboxy-terminal domain.

Regulation of m6A Transcripts by the 3'→5' RNA Helicase YTHDC2 Is Essential for a Successful Meiotic Program in the Mammalian Germline.

N6-methyladenosine (m6A) is an essential internal RNA modification that is critical for gene expression control in most organisms. Proteins with a YTH domain recognize m6A 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 m6A reader that is essential for male and female fertility in mice. High-throughput mapping of the m6A transcriptome and expression analysis in the Yhtdc2 mutant testes reveal an upregulation of m6A-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 m6A-modified germline transcripts to maintain a gene expression program that is conducive for progression through meiosis.

PIWI Slicing and EXD1 Drive Biogenesis of Nuclear piRNAs from Cytosolic Targets of the Mouse piRNA Pathway.

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.

Characterization and biological activities of melanin pigment from root endophytic fungus, Phoma sp. RDSE17.

Melanins are high molecular weight hydrophobic pigments which have gained popularity for their role in virulence against different pathogens. In the present study, we isolated and characterized the melanin pigment produced by a dark septate endophyte fungus Phoma sp. RDSE17, which was associated with the roots of an indigenous Oryza sativa cv. 'Chakhao amubi' in Manipur, Northeast India. The biological properties of purified melanin from the fungus were evaluated for their antioxidant, antimicrobial and anticancerous activities. The pigment was extracted from Phoma sp. by alkaline-acid hydrolysis method and confirmed as melanin through physico-chemical tests and spectral (UV, FTIR, and EPR) analysis. The analyses of the elemental composition indicated that the pigment possessed a low percentage of nitrogen (N) contents, and therefore, would not fall under DOPA class of melanin. Exposure of the fungus to melanin pathway inhibitors revealed a positive melanin inhibition by tricyclazole, but not by kojic acid. Thus, the melanin from Phoma sp. may be a member of the DHN family. Moreover, the purified melanin showed high DPPH (1, 1-Diphenyl-2-picrylhydrazyl) free radical-scavenging activity with an EC50 of 69 µg/mL and inhibited human lung cancer cell (A549 cells) proliferation at 80 µg/mL. The present study demonstrates that melanin from Phoma sp. RDSE17 could be employed as a potential biological (antioxidant) and antimicrobial agent for inhibiting the growth of humans and phytopathogens.

Recruitment of Armitage and Yb to a transcript triggers its phased processing into primary piRNAs in Drosophila ovaries.

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.

Primary piRNA biogenesis: caught up in a Maelstrom.

Precursors for most Piwi-interacting RNAs (piRNAs) are indistinguishable from other RNA polymerase II-transcribed long noncoding RNAs. So, it is currently unclear how they are recognized as substrates by the piRNA processing machinery that resides in cytoplasmic granules called nuage. In this issue, Castaneda et al (2014) reveal a role for the nuage component and nucleo-cytoplasmic shuttling protein Maelstrom in mouse piRNA biogenesis.

Metazoan Maelstrom is an RNA-binding protein that has evolved from an ancient nuclease active in protists.

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.

Mutations in the MOV10L1 ATP Hydrolysis Motif Cause piRNA Biogenesis Failure and Male Sterility in Mice.

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.

Kcnq1ot1 antisense noncoding RNA mediates lineage-specific transcriptional silencing through chromatin-level regulation.

Recent investigations have implicated long antisense noncoding RNAs in the epigenetic regulation of chromosomal domains. Here we show that Kcnq1ot1 is an RNA polymerase II-encoded, 91 kb-long, moderately stable nuclear transcript and that its stability is important for bidirectional silencing of genes in the Kcnq1 domain. Kcnq1ot1 interacts with chromatin and with the H3K9- and H3K27-specific histone methyltransferases G9a and the PRC2 complex in a lineage-specific manner. This interaction correlates with the presence of extended regions of chromatin enriched with H3K9me3 and H3K27me3 in the Kcnq1 domain in placenta, whereas fetal liver lacks both chromatin interactions and heterochromatin structures. In addition, the Kcnq1 domain is more often found in contact with the nucleolar compartment in placenta than in liver. Taken together, our data describe a mechanism whereby Kcnq1ot1 establishes lineage-specific transcriptional silencing patterns through recruitment of chromatin remodeling complexes and maintenance of these patterns through subsequent cell divisions occurs via targeting the associated regions to the perinucleolar compartment.

Tudor domain containing 12 (TDRD12) is essential for secondary PIWI interacting RNA biogenesis in mice.

Piwi-interacting RNAs (piRNAs) are gonad-specific small RNAs that provide defense against transposable genetic elements called transposons. Our knowledge of piRNA biogenesis is sketchy, partly due to an incomplete inventory of the factors involved. Here, we identify Tudor domain-containing 12 (TDRD12; also known as ECAT8) as a unique piRNA biogenesis factor in mice. TDRD12 is detected in complexes containing Piwi protein MILI (PIWIL2), its associated primary piRNAs, and TDRD1, all of which are already implicated in secondary piRNA biogenesis. Male mice carrying either a nonsense point mutation (reproductive mutant 23 or repro23 mice) or a targeted deletion in the Tdrd12 locus are infertile and derepress retrotransposons. We find that TDRD12 is dispensable for primary piRNA biogenesis but essential for production of secondary piRNAs that enter Piwi protein MIWI2 (PIWIL4). Cell-culture studies with the insect ortholog of TDRD12 suggest a role for the multidomain protein in mediating complex formation with other participants during secondary piRNA biogenesis.

Artificial Intelligence-Based Smart Comrade Robot for Elders Healthcare with Strait Rescue System.

A rising proportion of older people has more demand on services including hospitals, retirement homes, and assisted living facilities. Regaining control of this population's expectations will pose new difficulties for lawmakers, medical professionals, and the society at large. Smart technology can help older people to have independent and fulfilling lives while still living safely and securely in the community. In the last several decades, the number of sectors using robots has risen. Comrade robots have made their appearance in old human life, with the most recent notable appearance being in their care. The number of elderly individuals is increasing dramatically throughout the globe. The source of the story is the use of robots to help elderly people with day-to-day activities. Speech data and facial recognition model are done with AI model. Here, with the Comrade robotic model, elder people's healthcare system is designed with better analysis state. The aim is to put in place a simple robotic buddy to determine the health of the old person via a headband that has been given to them. Comrade robot may do things like senior citizens home automation, home equipment control, safety, and wellbeing sensing, and, in emergency situation, routine duties like navigating in the outside world. The fear that robotics and artificial intelligence would eventually eliminate most of the jobs is increasing. It is anticipated that, in order to survive and stay relevant in the constantly shifting environment of work, workers of the future will need to be creative and versatile and prepared to identify new business possibilities and change industry to meet challenges of the world. According to the research, reflective practice, time management, communicating, and collaboration are important in fostering creativity.

Transcriptional and Posttranscriptional Programming by Long Noncoding RNAs.

Recently, several lines of evidence have suggested that noncoding RNAs, which include both small and long noncoding RNAs (ncRNAs), contribute to a significant portion of the transcriptome in eukaryotic organisms. However, the functional significance of this wide-spread occurrence of ncRNAs, and in particular, the long ncRNAs (lncRNAs), for organismal development and differentiation is unclear. The available evidence from a subset of lncRNAs suggests that certain lncRNAs, and/or the act of their transcription, are involved in important biological functions at the transcriptional and posttranscriptional level. This chapter discusses the epigenetic and nonepigenetic mechanisms by which lncRNAs and/or their transcription are involved in the programming of various biological functions in model systems, from yeast to mammals.

The Mammalian Cap-Specific m6Am RNA Methyltransferase PCIF1 Regulates Transcript Levels in Mouse Tissues.

The 5' end of eukaryotic mRNAs is protected by the m7G-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 N6 position (m6A) by the mammalian Phosphorylated C-terminal domain (CTD)-interacting Factor 1 (PCIF1) to generate m6Am. Here, we show that although the loss of cap-specific m6Am 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 m6Am 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 m6Am methylase that contributes to the N6,N6,2'-O-trimethyladenosine (m62Am) in the hypermethylated cap4 structure of trypanosomatids. Thus, PCIF1 has evolved to function in catalytic and non-catalytic roles.

Exonuclease Domain-Containing 1 Enhances MIWI2 piRNA Biogenesis via Its Interaction with TDRD12.

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.