Beyond transcription: compelling open questions in plant RNA biology.

The study of RNAs has become one of the most influential research fields in contemporary biology and biomedicine. In the last few years, new sequencing technologies have produced an explosion of new and exciting discoveries in the field but have also given rise to many open questions. Defining these questions, together with old, long-standing gaps in our knowledge, is the spirit of this article. The breadth of topics within RNA biology research is vast, and every aspect of the biology of these molecules contains countless exciting open questions. Here, we asked 12 groups to discuss their most compelling question among some plant RNA biology topics. The following vignettes cover RNA alternative splicing; RNA dynamics; RNA translation; RNA structures; R-loops; epitranscriptomics; long non-coding RNAs; small RNA production and their functions in crops; small RNAs during gametogenesis and in cross-kingdom RNA interference; and RNA-directed DNA methylation. In each section, we will present the current state-of-the-art in plant RNA biology research before asking the questions that will surely motivate future discoveries in the field. We hope this article will spark a debate about the future perspective on RNA biology and provoke novel reflections in the reader.

Overcoming the field-of-view to diameter trade-off in microendoscopy via computational optrode-array microscopy.

High-resolution microscopy of deep tissue with large field-of-view (FOV) is critical for elucidating organization of cellular structures in plant biology. Microscopy with an implanted probe offers an effective solution. However, there exists a fundamental trade-off between the FOV and probe diameter arising from aberrations inherent in conventional imaging optics (typically, FOV < 30% of diameter). Here, we demonstrate the use of microfabricated non-imaging probes (optrodes) that when combined with a trained machine-learning algorithm is able to achieve FOV of 1x to 5x the probe diameter. Further increase in FOV is achieved by using multiple optrodes in parallel. With a 1 × 2 optrode array, we demonstrate imaging of fluorescent beads (including 30 FPS video), stained plant stem sections and stained living stems. Our demonstration lays the foundation for fast, high-resolution microscopy with large FOV in deep tissue via microfabricated non-imaging probes and advanced machine learning.

Arabidopsis mRNA decay landscape arises from specialized RNA decay substrates, decapping-mediated feedback, and redundancy.

The decay of mRNA plays a vital role in modulating mRNA abundance, which, in turn, influences cellular and organismal processes. In plants and metazoans, three distinct pathways carry out the decay of most cytoplasmic mRNAs: The mRNA decapping complex, which requires the scaffold protein VARICOSE (VCS), removes a protective 5' cap, allowing for 5' to 3' decay via EXORIBONUCLEASE4 (XRN4, XRN1 in metazoans and yeast), and both the exosome and SUPPRESSOR OF VCS (SOV)/DIS3L2 degrade RNAs in the 3' to 5' direction. However, the unique biological contributions of these three pathways, and whether they degrade specialized sets of transcripts, are unknown. In Arabidopsis, the participation of SOV in RNA homeostasis is also unclear, because Arabidopsis sov mutants have a normal phenotype. We carried out mRNA decay analyses in wild-type, sov, vcs, and vcs sov seedlings, and used a mathematical modeling approach to determine decay rates and quantify gene-specific contributions of VCS and SOV to decay. This analysis revealed that VCS (decapping) contributes to decay of 68% of the transcriptome, and, while it initiates degradation of mRNAs with a wide range of decay rates, it especially contributes to decay of short-lived RNAs. Only a few RNAs were clear SOV substrates in that they decayed more slowly in sov mutants. However, 4,506 RNAs showed VCS-dependent feedback in sov that modulated decay rates, and, by inference, transcription, to maintain RNA abundances, suggesting that these RNAs might also be SOV substrates. This feedback was shown to be independent of siRNA activity.

Noncanonical Alternative Polyadenylation Contributes to Gene Regulation in Response to Hypoxia.

Stresses from various environmental challenges continually confront plants, and their responses are important for growth and survival. One molecular response to such challenges involves the alternative polyadenylation of mRNA. In plants, it is unclear how stress affects the production and fate of alternative mRNA isoforms. Using a genome-scale approach, we show that in Arabidopsis thaliana, hypoxia leads to increases in the number of mRNA isoforms with polyadenylated 3' ends that map to 5'-untranslated regions (UTRs), introns, and protein-coding regions. RNAs with 3' ends within protein-coding regions and introns were less stable than mRNAs that end at 3'-UTR poly(A) sites. Additionally, these RNA isoforms were underrepresented in polysomes isolated from control and hypoxic plants. By contrast, mRNA isoforms with 3' ends that lie within annotated 5'-UTRs were overrepresented in polysomes and were as stable as canonical mRNA isoforms. These results indicate that the generation of noncanonical mRNA isoforms is an important feature of the abiotic stress response. The finding that several noncanonical mRNA isoforms are relatively unstable suggests that the production of non-stop and intronic mRNA isoforms may represent a form of negative regulation in plants, providing a conceptual link with mechanisms that generate these isoforms (such as alternative polyadenylation) and RNA surveillance.

Selective mRNA sequestration by OLIGOURIDYLATE-BINDING PROTEIN 1 contributes to translational control during hypoxia in Arabidopsis.

Low oxygen stress dynamically regulates the translation of cellular mRNAs as a means of energy conservation in seedlings of Arabidopsis thaliana. Most of the highly hypoxia-induced mRNAs are recruited to polysomes and actively translated, whereas other cellular mRNAs become translationally inactive and are either targeted for stabilization or degradation. Here we identify the involvement of OLIGOURIDYLATE BINDING PROTEIN 1 (UBP1), a triple RNA Recognition Motif protein, in dynamic and reversible aggregation of translationally repressed mRNAs during hypoxia. Mutation or down-regulation of UBP1C interferes with seedling establishment and reduces survival of low oxygen stress. By use of messenger ribonucleoprotein (mRNP) immunopurification, we show that UBP1C constitutively binds a subpopulation of mRNAs characterized by uracil-rich 3'-untranslated regions under normoxic conditions. During hypoxia, UBP1C association with non-uracil-rich mRNAs is enhanced concomitant with its aggregation into microscopically visible cytoplasmic foci, referred to as UBP1 stress granules (SGs). This UBP1C-mRNA association occurs as global levels of protein synthesis decline. Upon reoxygenation, rapid UBP1 SG disaggregation coincides with the return of the stabilized mRNAs to polysomes. The mRNAs that are highly induced and translated during hypoxia largely circumvent UBP1C sequestration. Thus, UBP1 is established as a component of dynamically assembled cytoplasmic mRNPs that sequester mRNAs that are poorly translated during a transient low energy stress.

Cold shock protein 1 chaperones mRNAs during translation in Arabidopsis thaliana.

RNA binding proteins (RBPs) function post-transcriptionally to fine-tune gene regulation. Arabidopsis thaliana has four Gly-rich, zinc finger-containing RBPs called cold shock proteins 1-4 (CSP1-CSP4), that possess an evolutionary conserved cold shock domain. Here, we determined that CSP1 associates with polyribosomes (polysomes) via an RNA-mediated interaction. Both the abundance and polysomal co-fractionation of CSP1 was enhanced in the cold (4°C), but did not influence global levels of polysomes, which were minimally perturbed by above freezing cold temperatures. Using a polyclonal antiserum, CSP1 was co-immunopurified with several hundred transcripts from rosettes of plants cultivated at 23°C or transferred to 4°C for 12 h. CSP1-associated mRNAs were characterized by G+C-rich 5' untranslated regions and gene ontologies related to cellular respiration, mRNA binding and translation. The majority of the CSP1-associated mRNAs were constitutively expressed and stable in the cold. CSP1 abundance was correlated with improved translation of ribosomal protein mRNAs during cold stress and improved maintenance of homeostasis and translation of mRNAs under water-deficit stress. In summary, CSP1 selectively chaperones mRNAs, providing translational enhancement during stress.

Health effects associated with exposure to intimate partner violence against women and childhood sexual abuse: a burden of proof study.

The health impacts of intimate partner violence against women and childhood sexual abuse are not fully understood. Here we conducted a systematic review by comprehensively searching seven electronic databases for literature on intimate partner violence-associated and childhood sexual abuse-associated health effects. Following the burden of proof methodology, we evaluated the evidence strength linking intimate partner violence and/or childhood sexual abuse to health outcomes supported by at least three studies. Results indicated a moderate association of intimate partner violence with major depressive disorder and with maternal abortion and miscarriage (63% and 35% increased risk, respectively). HIV/AIDS, anxiety disorders and self-harm exhibited weak associations with intimate partner violence. Fifteen outcomes were evaluated for their relationship to childhood sexual abuse, which was shown to be moderately associated with alcohol use disorders and with self-harm (45% and 35% increased risk, respectively). Associations between childhood sexual abuse and 11 additional health outcomes, such as asthma and type 2 diabetes mellitus, were found to be weak. Although our understanding remains limited by data scarcity, these health impacts are larger in magnitude and more extensive than previously reported. Renewed efforts on violence prevention and evidence-based approaches that promote healing and ensure access to care are necessary.

DHH1/DDX6-like RNA helicases maintain ephemeral half-lives of stress-response mRNAs.

Gene transcription is counterbalanced by messenger RNA decay processes that regulate transcript quality and quantity. We show here that the evolutionarily conserved DHH1/DDX6-like RNA hellicases of Arabidopsis thaliana control the ephemerality of a subset of cellular mRNAs. These RNA helicases co-localize with key markers of processing bodies and stress granules and contribute to their subcellular dynamics. They function to limit the precocious accumulation and ribosome association of stress-responsive mRNAs involved in auto-immunity and growth inhibition under non-stress conditions. Given the conservation of this RNA helicase subfamily, they may control basal levels of conditionally regulated mRNAs in diverse eukaryotes, accelerating responses without penalty.

Rapid immunopurification of ribonucleoprotein complexes of plants.

Hundreds of RNA binding proteins posttranscriptionally regulate gene expression, but relatively few have been characterized in plants. One successful approach to determine protein function has been to identify interacting molecules and the conditions of their association. The ribonucleoprotein immunopurification (RIP) assay facilitates the identification and quantitative comparison of RNA association to specific proteins under different experimental conditions. A variety of molecular techniques can be used to analyze the enriched RNAs, whether few as in the case of highly specific interactions, or many. Identification of associated RNAs can inform hypothesis generation about the processes or pathways regulated by the target protein. Downstream analysis of associated RNA sequences can lead to the identification of candidate motifs or features that mediate the protein-RNA interaction. We present a rapid method for RIP from tissues of plants that is suitable for experiments that require immediate tissue cryopreservation, such as monitoring a rapid response to an environmental stimulus.

Getting the message across: cytoplasmic ribonucleoprotein complexes.

mRNA-ribonucleoprotein (mRNP) complexes mediate post-transcriptional control mechanisms in the cell nucleus and cytoplasm. Transcriptional control is paramount to gene expression but is followed by regulated nuclear pre-mRNA maturation and quality control processes that culminate in the export of a functional transcript to the cytoplasm. Once in the cytosol, mRNPs determine the activity of individual mRNAs through regulation of localization, translation, sequestration and turnover. Here, we review how quantitative assessment of mRNAs in distinct cytoplasmic mRNPs, such as polyribosomes (polysomes), has provided new perspectives on post-transcriptional regulation from the global to gene-specific level. In addition, we explore recent genetic and biochemical studies of cytoplasmic mRNPs that have begun to expose RNA-binding proteins in an integrated network that fine-tunes gene expression.