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RNA-mediated chromatin silencing is central to genome regulation in many organisms. However, how nascent non-coding transcripts regulate chromatin is poorly understood. Here, through analysis of Arabidopsis FLC , we show that resolution of a nascent-transcript-induced R-loop promotes chromatin silencing. Stabilization of an antisense-induced R-loop at the 3′ end of FLC enables an RNA binding protein FCA, with its direct partner FY/WDR33 and other 3′-end processing factors, to polyadenylate the nascent antisense transcript. This clears the R-loop and recruits the chromatin modifiers demethylating H3K4me1. FCA immunoprecipitates with components of the m 6 A writer complex, and m 6 A modification affects dynamics of FCA nuclear condensates, and promotes FLC chromatin silencing. This mechanism also targets other loci in the Arabidopsis genome, and consistent with this fca and fy are hypersensitive to a DNA damage-inducing drug. These results show how modulation of R-loop stability by co-transcriptional RNA processing can trigger chromatin silencing. Nascent non-coding RNA can mediate chromatin silencing, however mechanistically this process is poorly understood. Here the authors show that resolution of an R-loop during 3'-end processing of a plant antisense transcript recruits chromatin modifiers to promote chromatin silencing.

RNA N 6 -methyladenosine (m 6 A) modifications are essential in plants. Here, we show that transgenic expression of the human RNA demethylase FTO in rice caused a more than threefold increase in grain yield under greenhouse conditions. In field trials, transgenic expression of FTO in rice and potato caused ~50% increases in yield and biomass. We demonstrate that the presence of FTO stimulates root meristem cell proliferation and tiller bud formation and promotes photosynthetic efficiency and drought tolerance but has no effect on mature cell size, shoot meristem cell proliferation, root diameter, plant height or ploidy. FTO mediates substantial m 6 A demethylation (around 7% of demethylation in poly(A) RNA and around 35% decrease of m 6 A in non-ribosomal nuclear RNA) in plant RNA, inducing chromatin openness and transcriptional activation. Therefore, modulation of plant RNA m 6 A methylation is a promising strategy to dramatically improve plant growth and crop yield. Rice and potato plants are more productive after epitranscriptome engineering.

Background N 6 -methyladenosine (m 6 A) mRNA modification is essential for mammalian and plant viability. The U6 m 6 A methyltransferases in other species regulate S-adenosylmethionine (SAM) homeostasis through installing m 6 A in pre-mRNAs of SAM synthetases. However, U6 m 6 A methyltransferase has not been characterized in Arabidopsis and little is known about its role in regulating photomorphogenesis and flowering. Results Here we characterize that FIONA1 is an Arabidopsis U6 m 6 A methyltransferase that installs m 6 A in U6 snRNA and a small subset of poly(A) + RNA. Disruption of FIONA1 leads to phytochrome signaling-dependent hypocotyl elongation and photoperiod-independent early flowering. Distinct from mammalian METTL16 and worm METT-10, FIONA1 neither installs m 6 A in the mRNAs of Arabidopsis SAM synthetases nor affects their transcript expression levels under normal or high SAM conditions. We confirm that FIONA1 can methylate plant mRNA m 6 A motifs in vitro and in vivo. We further show that FIONA1 installs m 6 A in several phenotypic related transcripts, thereby affecting downstream mRNA stability and regulating phytochrome signaling and floral transition. Conclusion FIONA1 is functional as a U6 m 6 A methyltransferase in Arabidopsis, distinct from mammalian METTL16 and worm METT-10. Our results demonstrate that FIONA1-mediated m 6 A post-transcriptional regulation is an autonomous regulator for flowering and phytochrome signaling-dependent photomorphogenesis.

As the most abundant and reversible chemical modification in eukaryotic mRNA, the epitranscriptomic mark N 6 -methyladenine (m 6 A) regulates plant development and stress response. We have previously characterized that ALKBH10B is an Arabidopsis mRNA m 6 A demethylase and regulates floral transition. However, it is unclear whether ALKBH10B plays a role in abiotic stress response. Here, we found that the expression of ALKBH10B is increased in response to abscisic acid (ABA), osmotic, and salt stress. The alkbh10b mutants showed hypersensitive to ABA, osmotic, and salt stress during seed germination. Transcriptome analysis revealed that the expression of several ABA response genes is upregulated in alkbh10b-1 than that of wild type, indicating ALKBH10B negatively affects the ABA signaling. Furthermore, m 6 A sequencing showed that ABA signaling genes, including PYR1 , PYL7 , PYL9 , ABI1 , and SnRK2.2 are m 6 A hypermethylated in alkbh10b-1 after ABA treatment. Taken together, our work demonstrated that ALKBH10B negatively modulates ABA response during seed germination in Arabidopsis.

Spermatogenesis is a differentiation process during which diploid spermatogonial stem cells （SSCs） produce hap- loid spermatozoa. This highly specialized process is precisely controlled at the transcriptional, posttranscriptional, and translational levels. Here we report that N6-methyladenosine （m6A）, an epitranscriptomic mark regulating gene expression, plays essential roles during spermatogenesis. We present comprehensive m6A mRNA methylomes of mouse spermatogenic cells from five developmental stages： undifferentiated spermatogonia, type At spermatogonia, preleptotene spermatocytes, pachytene/diplotene spermatocytes, and round spermatids. Germ cell-specific inactiva- tion of the m6A RNA methyltransferase Mettl3 or Mettll4 with Vasa-Cre causes loss of m6A and depletion of SSCs. m6A depletion dysregulates translation of transcripts that are required for SSC proliferation/differentiation. Com- bined deletion of Mettl3 and Mettll4 in advanced germ cells with Stra8-GFPCre disrupts spermiogenesis, whereas mice with single deletion of either Mettl3 or Mettll4 in advanced germ cells show normal spermatogenesis. The sper- matids from d6uble-mutant mice exhibit impaired translation of haploid-specific genes that are esseritial for spermio- genesis. This study highlights crucial roles of mRNA m6A modification in germline development, potentially ensuring coordinated translation at different stages of spermatogenesis.

Background RNA N 6 -methyladenosine (m 6 A) modification is critical for plant growth and crop yield. m 6 A reader proteins can recognize m 6 A modifications to facilitate the functions of m 6 A in gene regulation. ECT2, ECT3, and ECT4 are m 6 A readers that are known to redundantly regulate trichome branching and leaf growth, but their molecular functions remain unclear. Results Here, we show that ECT2, ECT3, and ECT4 directly interact with each other in the cytoplasm and perform genetically redundant functions in abscisic acid (ABA) response regulation during seed germination and post-germination growth. We reveal that ECT2/ECT3/ECT4 promote the stabilization of their targeted m 6 A-modified mRNAs, but have no function in alternative polyadenylation and translation. We find that ECT2 directly interacts with the poly(A) binding proteins, PAB2 and PAB4, and maintains the stabilization of m 6 A-modified mRNAs. Disruption of ECT2/ECT3/ECT4 destabilizes mRNAs of ABA signaling-related genes, thereby promoting the accumulation of ABI5 and leading to ABA hypersensitivity. Conclusion Our study reveals a unified functional model of m 6 A mediated by m 6 A readers in plants. In this model, ECT2/ECT3/ECT4 promote stabilization of their target mRNAs in the cytoplasm.

Recent years have witnessed rapid progress in the field of epitranscriptomics. Functional interpretation of the epitranscriptome relies on sequencing technologies that determine the location and stoichiometry of various RNA modifications. However, contradictory results have been reported among studies, bringing the biological impacts of certain RNA modifications into doubt. Here, we develop a synthetic RNA library resembling the endogenous transcriptome but without any RNA modification. By incorporating this modification-free RNA library into established mapping techniques as a negative control, we reveal abundant false positives resulting from sequence bias or RNA structure. After calibration, precise and quantitative mapping expands the understanding of two representative modification types, N6-methyladenosine (m6A) and 5-methylcytosine (m5C). We propose that this approach provides a systematic solution for the calibration of various RNA-modification mappings and holds great promise in epitranscriptomic studies.This work describes the generation of a modification-free RNA library that resembles endogenous transcriptome sequence and expression level, which can be used as a negative control in epitranscriptomic sequencing methods to obtain high-confidence and quantitative maps of various RNA modifications.

Certain adenosine residues within mammalian RNAs undergo reversible N 6 methylation. Two methyltransferase enzymes, METTL3 and METTL14, as well as the splicing factor WTAP are identified as core components of the multiprotein complex that deposits RNA N 6 -methyladenosine (m 6 A) in nuclear RNAs. N 6 -methyladenosine (m 6 A) is the most prevalent and reversible internal modification in mammalian messenger and noncoding RNAs. We report here that human methyltransferase-like 14 (METTL14) catalyzes m 6 A RNA methylation. Together with METTL3, the only previously known m 6 A methyltransferase, these two proteins form a stable heterodimer core complex of METTL3–METTL14 that functions in cellular m 6 A deposition on mammalian nuclear RNAs. WTAP, a mammalian splicing factor, can interact with this complex and affect this methylation.

N 6-Methyladenosine is an abundant nucleoside in cellular mRNA that undergoes demethylation under physiological conditions by fat mass and obesity-associated protein (FTO). This new pathway suggests that RNA modifications can be reversible and potentially have an impact on RNA metabolism. We report here that fat mass and obesity-associated protein (FTO) has efficient oxidative demethylation activity targeting the abundant N 6-methyladenosine (m 6 A) residues in RNA in vitro . FTO knockdown with siRNA led to increased amounts of m 6 A in mRNA, whereas overexpression of FTO resulted in decreased amounts of m 6 A in human cells. We further show the partial colocalization of FTO with nuclear speckles, which supports the notion that m 6 A in nuclear RNA is a major physiological substrate of FTO.

Profiling RNA modifications is essential to understand their functions and interactions. By taking the advantages of nanopore direct RNA sequencing, we present DirectRM, enabling simultaneous detection of six abundant modifications (N4-acetylcytidine, 1-methyladenosine, 5-methylcytidine, N7-methlguanosine, N6-methyladenosine, and pseudouridine) in native RNAs. Its two-stage pipeline identifies candidate modified kmers using binary classifier, then determines specific modifications and positions using an attention-based neural network. Trained with molecule-level features extracted from native RNA samples and validated on human cell lines and viral RNAs, DirectRM demonstrates high sensitivity, precision and robustness, outperforming existing tools. Crucially, we reveal the associations between modifications at both transcript and molecule-level. Modifications tend to proximate to each other on the transcript level, while at the molecule level, the presence of one modification is likely to reduce the occurrence of modifications at adjacent positions. DirectRM offers a powerful approach for studying epitranscriptome complexity and is expandable for future research. Profiling RNA modifications is essential to understanding their functions. Here, authors present DirectRM, a nanopore sequencing analytical tool that simultaneously detects six RNA modifications, revealing that modifications cluster at the transcript level but exclude each other on individual RNA molecules.