Specific recognition of N6-methyladenosine (m⁶A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The Arabidopsis YTH domain protein ECT2 is thought to bind to mRNA at URU(m⁶A)Y sites, yet RR(m⁶A)CH is the canonical m⁶A consensus site in all eukaryotes and ECT2 functions require m⁶A-binding activity. Here, we apply iCLIP (individual nucleotide resolution crosslinking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) to define high-quality target sets of ECT2 and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites. Our analyses show that ECT2 does in fact bind to RR(m⁶A)CH. Pyrimidine-rich motifs are enriched around, but not at m⁶A sites, reflecting a preference for N6-adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of m⁶A sites, are also implicated in ECT2 binding via its intrinsically disordered region (IDR). Finally, URUAY-type motifs are enriched at ECT2 crosslink sites, but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins. Our study provides coherence between genetic and molecular studies of m⁶A-YTH function in plants and reveals new insight into the mode of RNA recognition by YTH domain-containing proteins.

Gene regulation via N6-methyladenosine (m⁶A) in mRNA involves RNA-binding proteins that recognize m⁶A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only a few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant, ectopic, and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2, ECT3, and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady-state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.

Transcriptional regulation is tightly coupled with chromosomal positioning and three-dimensional chromatin architecture. However, it is unclear what proportion of transcriptional activity is reflecting such organisation, how much can be informed by RNA expression alone and how this impacts disease. Here, we develop a computational transcriptional decomposition approach separating the proportion of expression associated with genome organisation from independent effects not directly related to genomic positioning. We show that positionally attributable expression accounts for a considerable proportion of total levels and is highly informative of topological associating domain activities and organisation, revealing boundaries and chromatin compartments. Furthermore, expression data alone accurately predict individual enhancer–promoter interactions, drawing features from expression strength, stabilities, insulation and distance. We characterise predictions in 76 human cell types, observing extensive sharing of domains, yet highly cell-type-specific enhancer–promoter interactions and strong enrichments in relevant trait-associated variants. Overall, our work demonstrates a close relationship between transcription and chromatin architecture.

Mammalian gene promoters and enhancers share many properties. They are composed of a unified promoter architecture of divergent transcripton initiation and gene promoters may exhibit enhancer function. However, it is currently unclear how expression strength of a regulatory element relates to its enhancer strength and if the unifying architecture is conserved across Metazoa. Here we investigate the transcription initiation landscape and its associated RNA decay in Drosophila melanogaster. We find that the majority of active gene-distal enhancers and a considerable fraction of gene promoters are divergently transcribed. We observe quantitative relationships between enhancer potential, expression level and core promoter strength, providing an explanation for indirectly related histone modifications that are reflecting expression levels. Lowly abundant unstable RNAs initiated from weak core promoters are key characteristics of gene-distal developmental enhancers, while the housekeeping enhancer strengths of gene promoters reflect their expression strengths. The seemingly separable layer of regulation by gene promoters with housekeeping enhancer potential is also indicated by chromatin interaction data. Our results suggest a unified promoter architecture of many D. melanogaster regulatory elements, that is universal across Metazoa, whose regulatory functions seem to be related to their core promoter elements.

In virus-host interactions, nucleic acid-directed first lines of defense that allow viral clearance without compromising growth are of paramount importance. Plants use the RNA interference pathway as a basal antiviral immune system, but additional RNA-based mechanisms of defense also exist. The infectivity of a plant positive-strand RNA virus, alfalfa mosaic virus (AMV), relies on the demethylation of viral RNA by the recruitment of the cellular N6-methyladenosine (m⁶ A) demethylase ALKBH9B, but how demethylation of viral RNA promotes AMV infection remains unknown. Here, we show that inactivation of the Arabidopsis cytoplasmic YT521-B homology domain (YTH)-containing m⁶ A-binding proteins ECT2, ECT3, and ECT5 is sufficient to restore AMV infectivity in partially resistant alkbh9b mutants. We further show that the antiviral function of ECT2 is distinct from its previously demonstrated function in the promotion of primordial cell proliferation: an ect2 mutant carrying a small deletion in its intrinsically disordered region is partially compromised for antiviral defense but not for developmental functions. These results indicate that the m⁶ A-YTHDF axis constitutes a novel branch of basal antiviral immunity in plants.

Many genes are co‐expressed and form genomic domains of coordinated gene activity. However, the regulatory determinants of domain co‐activity remain unclear. Here, we leverage human individual variation in gene expression to characterize the co‐regulatory processes underlying domain co‐activity and systematically quantify their effect sizes. We employ transcriptional decomposition to extract from RNA expression data an expression component related to co‐activity revealed by genomic positioning. This strategy reveals close to 1,500 co‐activity domains, covering most expressed genes, of which the large majority are invariable across individuals. Focusing specifically on domains with high variability in co‐activity reveals that contained genes have a higher sharing of eQTLs, a higher variability in enhancer interactions, and an enrichment of binding by variably expressed transcription factors, compared to genes within non‐variable domains. Through careful quantification of the relative contributions of regulatory processes underlying co‐activity, we find transcription factor expression levels to be the main determinant of gene co‐activity. Our results indicate that distal trans effects contribute more than local genetic variation to individual variation in co‐activity domains.