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Adenosine bases of RNA can be transiently modified by the deposition of a methyl-group to form N 6 -methyladenosine (m 6 A). This adenosine-methylation is an ancient process and the enzymes involved are evolutionary highly conserved. A genetic screen designed to identify suppressors of late flowering transgenic Arabidopsis plants overexpressing the miP1a microProtein yielded a new allele of the FIONA1 (FIO1) m 6 A-methyltransferase. To characterize the early flowering phenotype of fio1 mutant plants we employed an integrative approach of mRNA-seq, Nanopore direct RNA-sequencing and meRIP-seq to identify differentially expressed transcripts as well as differentially methylated RNAs. We provide evidence that FIO1 is the elusive methyltransferase responsible for the 3’-end methylation of the FLOWERING LOCUS C ( FLC ) transcript. Furthermore, our genetic and biochemical data suggest that 3’-methylation stabilizes FLC mRNAs and non-methylated FLC is a target for rapid degradation.

An important component of cellular biochemistry is the concentration of proteins and nucleic acids in non-membranous compartments 1 , 2 . These biomolecular condensates are formed from processes that include liquid–liquid phase separation. The multivalent interactions necessary for liquid–liquid phase separation have been extensively studied in vitro 1 , 3 . However, the regulation of this process in vivo is poorly understood. Here we identify an in vivo regulator of liquid–liquid phase separation through a genetic screen targeting factors required for Arabidopsis RNA-binding protein FCA function. FCA contains prion-like domains that phase-separate in vitro, and exhibits behaviour in vivo that is consistent with phase separation. The mutant screen identified a functional requirement for FLL2, a coiled-coil protein, in the formation of FCA nuclear bodies. FCA reduces transcriptional read-through by promoting proximal polyadenylation at many sites in the Arabidopsis genome 3 , 4 . FLL2 was required to promote this proximal polyadenylation, but not the binding of FCA to target RNA. Ectopic expression of FLL2 increased the size and number of FCA nuclear bodies. Crosslinking with formaldehyde captured in vivo interactions between FLL2, FCA and the polymerase and nuclease modules of the RNA 3′-end processing machinery. These 3′ RNA-processing components colocalized with FCA in the nuclear bodies in vivo, which indicates that FCA nuclear bodies compartmentalize 3′-end processing factors to enhance polyadenylation at specific sites. Our findings show that coiled-coil proteins can promote liquid–liquid phase separation, which expands our understanding of the principles that govern the in vivo dynamics of liquid-like bodies. A genetic screen for factors required by the Arabidopsis RNA-binding protein FCA identifies FLL2 as necessary in the formation of FCA nuclear bodies, and thus a role for FLL2 in liquid–liquid phase separation.

Temperature-dependent alternative splicing of FLOWERING LOCUS M ( FLM ) results in two protein products, FLM-β and FLM-δ, that regulate the onset of flowering in Arabidopsis ; at cooler temperatures FLM-β represses flowering, whereas at higher temperatures, the plant preferentially produces FLM-δ, which promotes flowering. Flowering when the temperature is right The transition to flowering is a critical event in the life cycle of a flowering plant and it needs to be timed precisely to ensure reproductive success. Here Markus Schmid and colleagues study the regulation of flowering in response to changes in ambient temperature. They show that temperature-dependent alternative splicing of FLOWERING LOCUS M ( FLM ) yields two protein products, FLM-β and FLM-δ, that regulate flowering in opposite ways. At cooler temperatures FLM-β represses flowering, whereas at higher temperatures the plant preferentially produces FLM-δ to promote flowering. Hence, temperature-dependent alternative pre-mRNA splicing controls the onset of flowering. The appropriate timing of flowering is crucial for plant reproductive success. It is therefore not surprising that intricate genetic networks have evolved to perceive and integrate both endogenous and environmental signals, such as carbohydrate and hormonal status, photoperiod and temperature 1 , 2 . In contrast to our detailed understanding of the vernalization pathway, little is known about how flowering time is controlled in response to changes in the ambient growth temperature. In Arabidopsis thaliana , the MADS-box transcription factor genes FLOWERING LOCUS M ( FLM ) and SHORT VEGETATIVE PHASE ( SVP ) have key roles in this process 3 , 4 . FLM is subject to temperature-dependent alternative splicing 3 . Here we report that the two main FLM protein splice variants, FLM-β and FLM-δ, compete for interaction with the floral repressor SVP. The SVP–FLM-β complex is predominately formed at low temperatures and prevents precocious flowering. By contrast, the competing SVP–FLM-δ complex is impaired in DNA binding and acts as a dominant-negative activator of flowering at higher temperatures. Our results show a new mechanism that controls the timing of the floral transition in response to changes in ambient temperature. A better understanding of how temperature controls the molecular mechanisms of flowering will be important to cope with current changes in global climate 5 , 6 .

Understanding the regulation of fleshy fruit ripening is biologically important and provides insights and opportunities for controlling fruit quality, enhancing nutritional value for animals and humans, and improving storage and waste reduction. The ripening regulatory network involves master and downstream transcription factors (TFs) and hormones. Tomato is a model for ripening regulation, which requires ethylene and master TFs including NAC-NOR and the MADS-box protein MADS-RIN. Recent functional characterization showed that the classical RIN-MC gene fusion, previously believed to be a loss-of-function mutation, is an active TF with repressor activity. This, and other evidence, has highlighted the possibility that MADS-RIN itself is not important for ripening initiation but is required for full ripening. In this review, we discuss the diversity of components in the control network, their targets, and how they interact to control initiation and progression of ripening. Both hormones and individual TFs affect the status and activity of other network participants, which changes overall network signaling and ripening outcomes. MADS-RIN, NAC-NOR and ethylene play critical roles but there are still unanswered questions about these and other TFs. Further attention should be paid to relationships between ethylene, MADS-RIN and NACs in ripening control.

Long noncoding RNAs (IncRNAs) have been proposed to play important roles in gene regulation. However, their importance in epigenetic silencing and how specificity is determined remain controversial. We have investigated the cold-induced epigenetic switching mechanism involved in the silencing of Arabidopsis thaliana FLOWERING LOCUS C (FLC), which occurs during vernalization. Antisense transcripts, collectively named COOLAIR, are induced by prolonged cold before the major accumulation of histone 3 lysine 27 trimethylation (H3K27me3), characteristic of Polycomb silencing. We have found that COOLAIR is physically associated with the FLC locus and accelerates transcriptional shutdown of FLC during cold exposure. Removal of COOLAIR disrupted the synchronized replacement of H3K36 methylation with H3K27me3 at the intragenic FLC nucleation site during the cold. Consistently, genetic analysis showed COOLAIR and Polycomb complexes work independently in the cold-dependent silencing of FLC. Our data reveal a role for IncRNA in the coordinated switching of chromatin states that occurs during epigenetic regulation.

The appropriate timing of flowering is critical for plant reproductive success. Although the FLOWERING LOCUS T (FT)–FD module plays crucial roles in the photoperiodic flowering pathway, the underlying mechanisms and signaling pathways involved still remain elusive. Here, we demonstrate that class II TCP transcription factors (TFs) integrate into the FT–FD complex to control floral initiation in Arabidopsis (Arabidopsis thaliana). Class II CINCINNATA (CIN) TCP TFs function as transcriptional activators by directly binding to the promoters of downstream floral meristem identity genes, such as APETALA1 (AP1). In addition, these TCPs directly interact with FD, a basic Leu zipper TF that plays a critical role in photoperiodic flowering, which further activates AP1 expression. Genetic analyses indicated that class II CIN TCP TFs function synergistically with FT and FD, to positively regulate flowering in an AP1-dependent manner. Thus, our results provide compelling evidence that class II CIN TCP TFs act directly at the AP1 promoter to enhance its transcription, thus further elucidating the molecular mechanisms underlying the regulation of photoperiodic flowering in Arabidopsis.

Fruit ripening is a complex developmental process responsible for the transformation of the seed-containing organ into a tissue attractive to seed dispersers and agricultural consumers. The coordinated regulation of the different biochemical pathways necessary to achieve this change receives considerable research attention. The MADS-box transcription factor RIPENING INHIBITOR (RIN) is an essential regulator of tomato (Solarium lycopersicum) fruit ripening but the exact mechanism by which it influences the expression of ripening-related genes remains unclear. Using a chromatin immunoprecipitation approach, we provide evidence that RIN interacts with the promoters of genes involved in the major pathways associated with observed and well-studied ripening phenotypes and phenomena, including the transcriptional control network involved in overall ripening regulation, ethylene biosynthesis, ethylene perception, downstream ethylene response, cell wall metabolism, and carotenoid biosynthesis. Furthermore, in the cases of ethylene and carotenoid biosynthesis, RIN interacts with the promoters of genes encoding rate-limiting activities. We also show that RIN recruitment to target loci is dependent on a normally functioning alíele at the ripening-specific transcription factor COLORLESS NONRIPENING gene locus, further clarifying the relationship between these two ripening regulators.

Plants use seasonal temperature cues to time the transition to reproduction. In Arabidopsis thaliana , winter cold epigenetically silences the floral repressor locus FLOWERING LOCUS C ( FLC ) through POLYCOMB REPRESSIVE COMPLEX 2 (PRC2) 1 . This vernalization process aligns flowering with spring. A prerequisite for silencing is transcriptional downregulation of FLC , but how this occurs in the fluctuating temperature regimes of autumn is unknown 2 – 4 . Transcriptional repression correlates with decreased local levels of histone H3 trimethylation at K36 (H3K36me3) and H3 trimethylation at K4 (H3K4me3) 5 , 6 , which are deposited during FRIGIDA (FRI)-dependent activation of FLC 7 – 10 . Here we show that cold rapidly promotes the formation of FRI nuclear condensates that do not colocalize with an active FLC locus. This correlates with reduced FRI occupancy at the FLC promoter and FLC repression. Warm temperature spikes reverse this process, buffering FLC shutdown to prevent premature flowering. The accumulation of condensates in the cold is affected by specific co-transcriptional regulators and cold induction of a specific isoform of the antisense RNA COOLAIR 5 , 11 . Our work describes the dynamic partitioning of a transcriptional activator conferring plasticity in response to natural temperature fluctuations, thus enabling plants to effectively monitor seasonal progression. In Arabidopsis thaliana , downregulation of the floral repressor FLC in response to cold occurs through a mechanism in which the FLC activator FRIGIDA is sequestered into biomolecular condensates away from the FLC promoter.

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.

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2 , which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B , a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis. Histones modified in common lymphomas Despite being a focus of research activity for many years, the mutations driving the two most common non-Hodgkin lymphomas — follicular lymphoma and diffuse large B-cell lymphoma — have remained cryptic. Whole genome sequencing, combined with transcriptome analysis and further resequencing of candidate genes in additional tumours, now show that histone methyltransferases and acetylases are frequently affected by mutations in these tumours. This study suggests a previously unappreciated importance of chromatin biology in lymphomagenesis.