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 ) 1 . Different isoforms of COOLAIR influence FLC transcriptional output in warm and cold conditions 2 – 7 . 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 6 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.

In plants and animals, self-renewing stem cell populations play fundamental roles in many developmental contexts. Plants differ from most animals in their retained ability to initiate new cycles of growth and development, which relies on the establishment and activity of branch meristems. In seed plants, branching is achieved by stem-cell-containing axillary meristems, which are initiated from a leaf axil meristematic cell population originally detached from the shoot apical meristem. It remains unclear how the meristematic cell fate is maintained. Here, we show that ARABIDOPSISTHALIANAHOMEOBOXGENE1 (ATH1) maintains the meristem marker gene SHOOT MERISTEMLESS (STM) expression in the leaf axil to enable meristematic cell fate maintenance. Furthermore, ATH1 protein interacts with STM protein to form a STM self-activation loop. Genetic and biochemical data suggest that ATH1 anchors STM to activate STM as well as other axillary meristem regulatory genes. This auto-regulation allows the STM locus to remain epigenetically active. Taken together, our findings provide a striking example of a self-activation loop that maintains the flexibility required for stem cell niche re-establishment during organogenesis.

MicroRNA (miRNA)-mediated cleavage is involved in numerous essential cellular pathways. miRNAs recognize target RNAs via sequence complementarity. In addition to complementarity, in vitro and in silico studies have suggested that RNA structure may influence the accessibility of mRNAs to miRNA-induced silencing complexes (miRISCs), thereby affecting RNA silencing. However, the regulatory mechanism of mRNA structure in miRNA cleavage remains elusive. We investigated the role of in vivo RNA secondary structure in miRNA cleavage by developing the new CAP-STRUCTURE-seq method to capture the intact mRNA structurome in Arabidopsis thaliana. This approach revealed that miRNA target sites were not structurally accessible for miRISC binding prior to cleavage in vivo. Instead, we found that the unfolding of the target site structure plays a key role in miRISC activity in vivo. We found that the single-strandedness of the two nucleotides immediately downstream of the target site, named Target Adjacent nucleotide Motif, can promote miRNA cleavage but not miRNA binding, thus decoupling target site binding from cleavage. Our findings demonstrate that mRNA structure in vivo can modulate miRNA cleavage, providing evidence of mRNA structure-dependent regulation of biological processes.

Abstract 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.

RNA secondary structure plays a critical role in gene regulation. Rice (Otyza sativa) is one of the most important food crops in the world. However, RNA structure in rice has scarcely been studied. Here, we have successfully generated in vivo Structure-seq libraries in rice. We found that the structural flexibility of mRNAs might associate with the dynamics of biological function. Higher N-6-methyladenosine (m(6)A) modification tends to have less RNA structure in 3' UTR, whereas GC content does not significantly affect in vivo mRNA structure to maintain efficient biological processes such as translation. Comparative analysis of RNA structurome between rice and Arabidopsis revealed that higher GC content does not lead to stronger structure and less RNA structural flexibility. Moreover, we found a weak correlation between sequence and structure conservation of the orthologs between rice and Arabidopsis. The conservation and divergence of both sequence and in vivo RNA structure corresponds to diverse and specific biological processes. Our results indicate that RNA secondary structure might offer a separate layer of selection to the sequence between monocot and dicot. Therefore, our study implies that RNA structure evolves differently in various biological processes to maintain robustness in development and adaptational flexibility during angiosperm evolution.

The dynamic structure of RNA plays a central role in post-transcriptional regulation of gene expression such as RNA maturation, degradation, and translation. With the rise of next-generation sequencing, the study of RNA structure has been transformed from in vitro low-throughput RNA structure probing methods to in vivo high-throughput RNA structure profiling. The development of these methods enables incremental studies on the function of RNA structure to be performed, revealing new insights of novel regulatory mechanisms of RNA structure in plants. Genome-wide scale RNA structure profiling allows us to investigate general RNA structural features over 10s of 1000s of mRNAs and to compare RNA structuromes between plant species. Here, we provide a comprehensive and up-to-date overview of: (i) RNA structure probing methods; (ii) the biological functions of RNA structure; (iii) genome-wide RNA structural features corresponding to their regulatory mechanisms; and (iv) RNA structurome evolution in plants.

One distinctive feature of plant post-embryonic development is that plants can undergo reiterative growth and continuous organogenesis throughout their lifetimes. Axillary meristems (AMs) in leaf axils play a central role in this growth and differences in meristem initiation and development produce the diversity of plant architecture. Studies in the past 15 years have shown that several transcription factors (TFs) and phytohormones affect AM initiation. In this review, we highlight recent research using systems biology approaches to examine the regulatory hierarchies underlying AM initiation and the role of auxins and cytokinins in AM initiation and development. This research revealed a developmental mechanism in which phytohormone signals act with a gene regulatory network containing multiple TFs to contribute to the initiation of AMs.

Pistacia chinensis Bunge (P. chinensis) is a deciduous and dioecious perennial arbor of the family Anacardiaceae that flowers from March to April and bears fruit from September to October. There are three rapidly growing stages in the annual growth process of P. chinensis. However, the knowledge of the secondary metabolites related to P. chinensis gender and growth season remains scant. In this study, HPLC was used to qualitatively and quantitatively determine the content of the catechin hydrate, rutin, quercetin, and kaempferol contents in male and female tree inflorescences and leaves. Total phenolics and flavonoids were also detected using a spectrophotometer. The results indicated that the contents of these compounds fluctuated with seasons and they reached the highest levels in nascent leaves. The fluctuations of these compounds followed different pathways of evolution, by increasing or decreasing in male and female trees throughout the whole growth process because they had their own biological functions. Moreover, the extracts exhibited DPPH radical scavenging bioactivity and showed no significant cytotoxicity towards 3T3-L1 preadipocytes. Together, these results demonstrated that P. chinensis has great potential as an antioxidant medicine, and the best harvest time is in the spring.

An efficient enzyme-responsive controlled release carrier system was successfully fabricated using single-stranded DNA encapsulated functional mesoporous silica nanoparticles. Mesoporous silica nanoparticles were initially fabricated through hydrolysis of tetraethyl orthosilicate (TEOS) in cetyltrimethylammonium chloride (CTAC) solution, and the surface of nanoparticles could be encapsulated with single-stranded DNA. This nanomaterial has excellent bioactivity and its hydrolysate cannot cause damage to normal cell, thus the biocompatibility of the nanomaterial is improved. In addition, this nanomaterial showed an excellent drug release performance when loaded with drugs, which would be helpful to increase the treatment efficiency and decrease side effects of drugs.