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Nucleotide composition is suggested to infer gene functionality and ecological adaptation of species to distinct environments. However, the underlying biological function of nucleotide composition dictating environmental adaptations is largely unknown. Here, we systematically analyze the nucleotide composition of transcriptomes across 1000 plants (1KP) and their corresponding habitats. Intriguingly, we find that plants growing in cold climates have guanine (G)-enriched transcriptomes, which are prone to forming RNA G-quadruplex structures. Both immunofluorescence detection and in vivo structure profiling reveal that RNA G-quadruplex formation in plants is globally enhanced in response to cold. Cold-responsive RNA G-quadruplexes strongly enhanced mRNA stability, rather than affecting translation. Disruption of individual RNA G-quadruplex promotes mRNA decay in the cold, leading to impaired plant cold response. Therefore, we propose that plants adopted RNA G-quadruplex structure as a molecular signature to facilitate their adaptation to the cold during evolution.

RNA G-quadruplex (rG4) is a vital RNA tertiary structure motif that involves the base pairs on both Hoogsteen and Watson-Crick faces of guanines. rG4 is of great importance in the post-transcriptional regulation of gene expression. Experimental technologies have advanced to identify in vitro and in vivo rG4s across diverse transcriptomes. Building on these recent advances, here we present G4Atlas, the first transcriptome-wide G-quadruplex database, in which we have collated, classified, and visualized transcriptome rG4 experimental data, generated from rG4-seq, chemical profiling and ligand-binding methods. Our comprehensive database includes transcriptome-wide rG4s generated from 82 experimental treatments and 238 samples across ten species. In addition, we have also included RNA secondary structure prediction information across both experimentally identified and unidentified rG4s to enable users to display any potential competitive folding between rG4 and RNA secondary structures. As such, G4Atlas will enable users to explore the general functions of rG4s in diverse biological processes. In addition, G4Atlas lays the foundation for further data-driven deep learning algorithms to examine rG4 structural features.

Cellular RNAs are heterogeneous with respect to their alternative processing and secondary structures, but the functional importance of this complexity is still poorly understood. A set of alternatively processed antisense non-coding transcripts, which are collectively called COOLAIR, are generated at the Arabidopsis floral-repressor locus FLOWERING LOCUS C (FLC). Different isoforms of COOLAIR influence FLC transcriptional output in warm and cold conditions. Here, to further investigate the function of COOLAIR, we developed an RNA structure-profiling method to determine the in vivo structure of single RNA molecules rather than the RNA population average. This revealed that individual isoforms of the COOLAIR transcript adopt multiple structures with different conformational dynamics. The major distally polyadenylated COOLAIR isoform in warm conditions adopts three predominant structural conformations, the proportions and conformations of which change after cold exposure. An alternatively spliced, strongly cold-upregulated distal COOLAIR isoform shows high structural diversity, in contrast to proximally polyadenylated COOLAIR. A hyper-variable COOLAIR structural element was identified that was complementary to the FLC transcription start site. Mutations altering the structure of this region changed FLC expression and flowering time, consistent with an important regulatory role of the COOLAIR structure in FLC transcription. Our work demonstrates that isoforms of non-coding RNA transcripts adopt multiple distinct and functionally relevant structural conformations, which change in abundance and shape in response to external conditions.

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Liquid-liquid phase separation plays an important role in a variety of cellular processes, including the formation of membrane-less organelles, the cytoskeleton, signalling complexes, and many other biological supramolecular assemblies. Studies on the molecular basis of phase separation in cells have focused on protein-driven phase separation. In contrast, there is limited understanding on how RNA specifically contributes to phase separation. Here, we described a phase-separation-like phenomenon that SHORT ROOT (SHR) RNA undergoes in cells. We found that an RNA G-quadruplex (GQ) forms in SHR mRNA and is capable of triggering RNA phase separation under physiological conditions, suggesting that GQs might be responsible for the formation of the SHR phase-separation-like phenomenon in vivo. We also found the extent of GQ-triggered-phase-separation increases on exposure to conditions which promote GQ. Furthermore, GQs with more G-quartets and longer loops are more likely to form phase separation. Our studies provide the first evidence that RNA can adopt structural motifs to trigger and/or maintain the specificity of RNA-driven phase separation.

Low temperatures are required to regulate the transition from vegetative to reproductive growth via a pathway called vernalization. In wheat, vernalization predominantly involves the cold upregulation of the floral activator (). Here, we have used an extreme vernalization response, identified through studying ambient temperature responses, to reveal the complexity of temperature inputs into , with allelic inter-copy variation at a gene expansion of modulating these effects. We find that the repressors of the reproductive transition, () and , are re-activated when plants experience high temperatures during and after vernalization. In addition, this re-activation is regulated by photoperiod for but was independent of photoperiod for We also find this warm temperature interruption affects flowering time and floret number and is stage specific. This research highlights the important balance between floral activators and repressors in coordinating the response of a plant to temperature, and that the absence of warmth is essential for the completion of vernalization. This knowledge can be used to develop agricultural germplasm with more predictable vernalization responses that will be more resilient to variable growth temperatures.

Salicylic acid (SA) is a plant defense hormone required for immunity. Arabidopsis NPR1 and NPR3/NPR4 were previously shown to bind SA and all three proteins were proposed as SA receptors. NPR1 functions as a transcriptional co-activator, whereas NPR3/NPR4 were suggested to function as E3 ligases that promote NPR1 degradation. Here we report that NPR3/NPR4 function as transcriptional co-repressors and SA inhibits their activities to promote the expression of downstream immune regulators. npr4-4D, a gain-of-function npr4 allele that renders NPR4 unable to bind SA, constitutively represses SA-induced immune responses. In contrast, the equivalent mutation in NPR1 abolishes its ability to bind SA and promote SA-induced defense gene expression. Further analysis revealed that NPR3/NPR4 and NPR1 function independently to regulate SA-induced immune responses. Our study indicates that both NPR1 and NPR3/NPR4 are bona fide SA receptors, but play opposite roles in transcriptional regulation of SA-induced defense gene expression.