RESUMO
The development of high-throughput RNA structure profiling methods in the past decade has greatly facilitated our ability to map and characterize different aspects of RNA structures transcriptome-wide in cell populations, single cells and single molecules. The resulting high-resolution data have provided insights into the static and dynamic nature of RNA structures, revealing their complexity as they perform their respective functions in the cell. In this Review, we discuss recent technical advances in the determination of RNA structures, and the roles of RNA structures in RNA biogenesis and functions, including in transcription, processing, translation, degradation, localization and RNA structure-dependent condensates. We also discuss the current understanding of how RNA structures could guide drug design for treating genetic diseases and battling pathogenic viruses, and highlight existing challenges and future directions in RNA structure research.
RESUMO
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 conditions2-7. 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 isoform6 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.
Assuntos
Arabidopsis , Conformação de Ácido Nucleico , RNA Antissenso , RNA de Plantas , RNA não Traduzido , Imagem Individual de Molécula , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Flores/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Domínio MADS/genética , RNA Antissenso/química , RNA Antissenso/genética , RNA de Plantas/química , RNA de Plantas/genética , RNA não Traduzido/química , RNA não Traduzido/genética , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
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.
Assuntos
Regulação da Expressão Gênica , RNA , RNA de Plantas/genética , RNA/genética , Interferência de RNA , Metilação , BiologiaRESUMO
i-Motifs (iMs), are secondary structures formed in cytosine-rich DNA sequences and are involved in multiple functions in the genome. Although putative iM forming sequences are widely distributed in the human genome, the folding status and strength of putative iMs vary dramatically. Much previous research on iM has focused on assessing the iM folding properties using biophysical experiments. However, there are no dedicated computational tools for predicting the folding status and strength of iM structures. Here, we introduce a machine learning pipeline, iM-Seeker, to predict both folding status and structural stability of DNA iMs. The programme iM-Seeker incorporates a Balanced Random Forest classifier trained on genome-wide iMab antibody-based CUT&Tag sequencing data to predict the folding status and an Extreme Gradient Boosting regressor to estimate the folding strength according to both literature biophysical data and our in-house biophysical experiments. iM-Seeker predicts DNA iM folding status with a classification accuracy of 81% and estimates the folding strength with coefficient of determination (R2) of 0.642 on the test set. Model interpretation confirms that the nucleotide composition of the C-rich sequence significantly affects iM stability, with a positive correlation with sequences containing cytosine and thymine and a negative correlation with guanine and adenine.
Assuntos
DNA , Aprendizado de Máquina , Motivos de Nucleotídeos , Humanos , Sequência de Bases , Citosina/química , DNA/química , DNA/genéticaRESUMO
DNA, beyond its canonical B-form double helix, adopts various alternative conformations, among which the i-motif, emerging in cytosine-rich sequences under acidic conditions, holds significant biological implications in transcription modulation and telomere biology. Despite recognizing the crucial role of i-motifs, predictive software for i-motif forming sequences has been limited. Addressing this gap, we introduce 'iM-Seeker', an innovative computational platform designed for the prediction and evaluation of i-motifs. iM-Seeker exhibits the capability to identify potential i-motifs within DNA segments or entire genomes, calculating stability scores for each predicted i-motif based on parameters such as the cytosine tracts number, loop lengths, and sequence composition. Furthermore, the webserver leverages automated machine learning (AutoML) to effortlessly fine-tune the optimal i-motif scoring model, incorporating user-supplied experimental data and customised features. As an advanced, versatile approach, 'iM-Seeker' promises to advance genomic research, highlighting the potential of i-motifs in cell biology and therapeutic applications. The webserver is freely available at https://im-seeker.org.
Assuntos
DNA , Internet , Aprendizado de Máquina , Motivos de Nucleotídeos , Software , DNA/química , DNA/genética , Humanos , Análise de Sequência de DNA/métodos , AlgoritmosRESUMO
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.
Assuntos
Quadruplex G , Transcriptoma , Algoritmos , Guanina , RNA/genética , RNA/química , Transcriptoma/genética , Atlas como AssuntoRESUMO
Subcellular mRNA quantities and spatial distributions are fundamental for driving gene regulatory programmes. Single molecule RNA fluorescence in situ hybridization (smFISH) uses fluorescent probes to label individual mRNA molecules, thereby facilitating both localization and quantitative studies. Validated reference mRNAs function as positive controls and are required for calibration. Here we present selection criteria for the first set of Arabidopsis smFISH reference genes. Following sequence and transcript data assessments, four mRNA probe sets were selected for imaging. Transcript counts per cell, correlations with cell size, and corrected fluorescence intensities were all calculated for comparison. In addition to validating reference probe sets, we present sample preparation steps that can retain green fluorescent protein fluorescence, thereby providing a method for simultaneous RNA and protein detection. In summary, our reference gene analyses, modified protocol, and simplified quantification method together provide a firm foundation for future quantitative single molecule RNA studies in Arabidopsis root apical meristem cells.
Assuntos
Arabidopsis , RNA , RNA/genética , Arabidopsis/genética , Hibridização in Situ Fluorescente/métodos , RNA Mensageiro/genética , Regulação da Expressão GênicaRESUMO
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 VERNALIZATION1 (VRN1). Here, we have used an extreme vernalization response, identified through studying ambient temperature responses, to reveal the complexity of temperature inputs into VRN-A1, with allelic inter-copy variation at a gene expansion of VRN-A1 modulating these effects. We find that the repressors of the reproductive transition, VERNALIZATION2 (VRN2) and ODDSOC2, are re-activated when plants experience high temperatures during and after vernalization. In addition, this re-activation is regulated by photoperiod for VRN2 but was independent of photoperiod for ODDSOC2 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.
Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Temperatura Alta , Fotoperíodo , Proteínas de Plantas/metabolismo , Proteínas Repressoras/metabolismo , Triticum/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas Repressoras/genética , Triticum/genéticaRESUMO
RNA structural elements occur in numerous single-stranded positive-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' untranslated region (UTR) in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts imply a key role for s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodeled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as antiviral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses, including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The antiviral potential of this element represents a promising start for further research into targeting conserved elements in RNA viruses.
Assuntos
COVID-19 , Genoma Viral , Motivos de Nucleotídeos , Dobramento de RNA , RNA Viral , SARS-CoV-2/fisiologia , Replicação Viral , Animais , COVID-19/genética , COVID-19/metabolismo , Chlorocebus aethiops , Células HEK293 , Humanos , RNA Viral/genética , RNA Viral/metabolismo , Células VeroRESUMO
Most plant pentatricopeptide repeat (PPR) proteins localize to and function inside plastids and mitochondria. However, the function of PPRs that only localize to the cytoplasm remains unknown. Here, we demonstrated that the rice (Oryza sativa) PPR protein CYTOPLASM-LOCALIZED PPR1 (OsCPPR1) contributes to pollen development and localizes to the cytoplasm. Knocking down OsCPPR1 led to abnormal plastid development in tapetal cells, prolonged tapetal programmed cell death (PCD) and tapetum degradation, and significantly reduced pollen fertility. Transcriptome analysis revealed that the transcript level of OsGOLDEN-LIKE1 (OsGLK1), which encodes a transcription factor that regulates plastid development and maintenance, was significantly higher in the OsCPPR1 knockdown plants compared to wild-type plants. We further determined that OsCPPR1 downregulates OsGLK1 transcription by directly binding to the single-stranded regions of OsGLK1 mRNAs. Overexpression of OsGLK1 resulted in abnormal tapetum and plastid development, similar to that seen in OsCPPR1 knockdown plants, and suppression of OsGLK1 partially restored pollen fertility in the OsCPPR1 knockdown plants. We therefore conclude that OsCPPR1 suppresses OsGLK1 in the regulation of plastid development and PCD in the tapetum. Our work revealed novel functions for a cytosolic PPR, demonstrating the diverse roles of PPRs in plants and identifying a new regulatory mechanism for regulating pollen development in rice.
Assuntos
Oryza , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plastídeos/genética , Plastídeos/metabolismo , PólenRESUMO
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.
Assuntos
MicroRNAs/ultraestrutura , Conformação de Ácido Nucleico , Interferência de RNA , RNA/ultraestrutura , Arabidopsis/genética , Sítios de Ligação/genética , MicroRNAs/genética , RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/genética , RNA Mensageiro/genética , Complexo de Inativação Induzido por RNA/genéticaRESUMO
RNA folding is an intrinsic property of RNA that serves a key role in every step of post-transcriptional regulation of gene expression, from RNA maturation to translation in plants. Recent developments of genome-wide RNA structure profiling methods have transformed research in this area enabling focus to shift from individual molecules to the study of tens of thousands of RNAs. Here, we provide a comprehensive review of recent advances in the field. We discuss these new insights of RNA structure functionality within the context of post-transcriptional regulation including mRNA maturation, translation, and RNA degradation in plants. Notably, we also provide an overview of how plants exhibit different RNA structures in response to environmental changes.
Assuntos
Regulação da Expressão Gênica de Plantas , Conformação de Ácido Nucleico , Plantas/química , RNA de Plantas/genética , Transcrição Gênica , Plantas/genética , RNA de Plantas/químicaRESUMO
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.
Assuntos
Quadruplex G , Transição de Fase , RNA/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Extração Líquido-Líquido , Conformação de Ácido Nucleico , Raízes de Plantas/química , RNA/isolamento & purificação , RNA/fisiologia , RNA Mensageiro/química , RNA Mensageiro/isolamento & purificação , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
RNA structure has critical roles in processes ranging from ligand sensing to the regulation of translation, polyadenylation and splicing. However, a lack of genome-wide in vivo RNA structural data has limited our understanding of how RNA structure regulates gene expression in living cells. Here we present a high-throughput, genome-wide in vivo RNA structure probing method, structure-seq, in which dimethyl sulphate methylation of unprotected adenines and cytosines is identified by next-generation sequencing. Application of this method to Arabidopsis thaliana seedlings yielded the first in vivo genome-wide RNA structure map at nucleotide resolution for any organism, with quantitative structural information across more than 10,000 transcripts. Our analysis reveals a three-nucleotide periodic repeat pattern in the structure of coding regions, as well as a less-structured region immediately upstream of the start codon, and shows that these features are strongly correlated with translation efficiency. We also find patterns of strong and weak secondary structure at sites of alternative polyadenylation, as well as strong secondary structure at 5' splice sites that correlates with unspliced events. Notably, in vivo structures of messenger RNAs annotated for stress responses are poorly predicted in silico, whereas mRNA structures of genes related to cell function maintenance are well predicted. Global comparison of several structural features between these two categories shows that the mRNAs associated with stress responses tend to have more single-strandedness, longer maximal loop length and higher free energy per nucleotide, features that may allow these RNAs to undergo conformational changes in response to environmental conditions. Structure-seq allows the RNA structurome and its biological roles to be interrogated on a genome-wide scale and should be applicable to any organism.
Assuntos
Arabidopsis/genética , Genoma de Planta/genética , Conformação de Ácido Nucleico , RNA de Plantas/química , RNA de Plantas/metabolismo , Sequências Reguladoras de Ácido Ribonucleico , Sequência de Bases , Códon de Iniciação/genética , Biologia Computacional , Dados de Sequência Molecular , Filogenia , Poliadenilação/genética , Biossíntese de Proteínas/genética , Sítios de Splice de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/análise , RNA de Plantas/genética , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Sequências Reguladoras de Ácido Ribonucleico/genética , Análise de Sequência de RNA , Estresse Fisiológico/genética , Relação Estrutura-AtividadeRESUMO
Most RNA molecules form internal base pairs, leading to a folded secondary structure. Some of these structures have been demonstrated to be functionally significant. High-throughput RNA structure chemical probing methods generate millions of sequencing reads to provide structural constraints for RNA secondary structure prediction. At present, processed data from these experiments are difficult to access without computational expertise. Here we present FoldAtlas, a web interface for accessing raw and processed structural data across thousands of transcripts. FoldAtlas allows a researcher to easily locate, view, and retrieve probing data for a given RNA molecule. We also provide in silico and in vivo secondary structure predictions for comparison, visualized in the browser as circle plots and topology diagrams. Data currently integrated into FoldAtlas are from a new high-depth Structure-seq data analysis in Arabidopsis thaliana, released with this work. AVAILABILITY AND IMPLEMENTATION: The FoldAtlas website can be accessed at www.foldatlas.com Source code is freely available at github.com/mnori/foldatlas under the MIT license. Raw reads data are available under the NCBI SRA accession SRP066985. CONTACT: yiliang.ding@jic.ac.uk or matthew.norris@jic.ac.ukSupplementary information: Supplementary data are available at Bioinformatics online.
Assuntos
Bases de Dados de Ácidos Nucleicos , RNA/metabolismo , Arabidopsis/metabolismo , Simulação por Computador , Conformação de Ácido Nucleico , RNA/química , RNA de Plantas/química , RNA de Plantas/metabolismo , Análise de Sequência de RNARESUMO
Biological nitrogen fixation in legumes occurs in nodules that are initiated in the root cortex following Nod factor recognition at the root surface, and this requires coordination of diverse developmental programs in these different tissues. We show that while early Nod factor signaling associated with calcium oscillations is limited to the root surface, the resultant activation of Nodule Inception (NIN) in the root epidermis is sufficient to promote cytokinin signaling and nodule organogenesis in the inner root cortex. NIN or a product of its action must be associated with the transmission of a signal between the root surface and the cortical cells where nodule organogenesis is initiated. NIN appears to have distinct functions in the root epidermis and the root cortex. In the epidermis, NIN restricts the extent of Early Nodulin 11 (ENOD11) expression and does so through competitive inhibition of ERF Required for Nodulation (ERN1). In contrast, NIN is sufficient to promote the expression of the cytokinin receptor Cytokinin Response 1 (CRE1), which is restricted to the root cortex. Our work in Medicago truncatula highlights the complexity of NIN action and places NIN as a central player in the coordination of the symbiotic developmental programs occurring in differing tissues of the root that combined are necessary for a nitrogen-fixing symbiosis.
Assuntos
Medicago truncatula/genética , Proteínas de Plantas/metabolismo , Transdução de Sinais , Sinorhizobium meliloti/fisiologia , Simbiose , Fatores de Transcrição/metabolismo , Cálcio/metabolismo , Citocininas/metabolismo , Regulação da Expressão Gênica de Plantas , Genes Reporter , Medicago truncatula/citologia , Medicago truncatula/fisiologia , Fixação de Nitrogênio , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Nodulação , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas , Nódulos Radiculares de Plantas/citologia , Nódulos Radiculares de Plantas/genética , Nódulos Radiculares de Plantas/fisiologia , Nicotiana/citologia , Nicotiana/genética , Nicotiana/fisiologia , Fatores de Transcrição/genéticaRESUMO
Plant roots have the potential capacity to grow almost indefinitely if meristematic and lateral branching is sustained. In a genetic screen we identified an Arabidopsis mutant showing limited root growth (lrg1) due to defects in cell division and elongation in the root meristem. Positional cloning determined that lrg1 affects an alpha-1,2-mannosyltransferase gene, LEW3, involved in protein N-glycosylation. The lrg1 mutation causes a synonymous substitution that alters the correct splicing of the fourth intron in LEW3, causing a mix of wild-type and truncated protein. LRG1 RNA missplicing in roots and short root phenotypes in lrg1 are light-intensity dependent. This mutation disrupts a GC-base pair in a three-base-pair stem with a four-nucleotide loop, which seems to be necessary for correct LEW3 RNA splicing. We found that the lrg1 short root phenotype correlates with high levels of reactive oxygen species and low pH in the apoplast. Proteomic analyses of N-glycosylated proteins identified GLU23/PYK10 and PRX34 as N-glycosylation targets of LRG1 activity. The lrg1 mutation reduces the positive interaction between Arabidopsis and Serendipita indica. A prx34 mutant showed a significant reduction in root growth, which is additive to lrg1. Taken together our work highlights the important role of N-glycosylation in root growth and development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Basidiomycota/fisiologia , Manosiltransferases/metabolismo , Peroxidases/metabolismo , beta-Glucosidase/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Divisão Celular , Glicosilação , Concentração de Íons de Hidrogênio , Íntrons/genética , Manosiltransferases/genética , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/efeitos da radiação , Mutação , Peroxidases/genética , Fenótipo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/efeitos da radiação , Proteômica , Splicing de RNA , Espécies Reativas de Oxigênio/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/efeitos da radiação , beta-Glucosidase/genéticaRESUMO
MOTIVATION: RNAs fold into complex structures that are integral to the diverse mechanisms underlying RNA regulation of gene expression. Recent development of transcriptome-wide RNA structure profiling through the application of structure-probing enzymes or chemicals combined with high-throughput sequencing has opened a new field that greatly expands the amount of in vitro and in vivo RNA structural information available. The resultant datasets provide the opportunity to investigate RNA structural information on a global scale. However, the analysis of high-throughput RNA structure profiling data requires considerable computational effort and expertise. RESULTS: We present a new platform, StructureFold, that provides an integrated computational solution designed specifically for large-scale RNA structure mapping and reconstruction across any transcriptome. StructureFold automates the processing and analysis of raw high-throughput RNA structure profiling data, allowing the seamless incorporation of wet-bench structural information from chemical probes and/or ribonucleases to restrain RNA secondary structure prediction via the RNAstructure and ViennaRNA package algorithms. StructureFold performs reads mapping and alignment, normalization and reactivity derivation, and RNA structure prediction in a single user-friendly web interface or via local installation. The variation in transcript abundance and length that prevails in living cells and consequently causes variation in the counts of structure-probing events between transcripts is accounted for. Accordingly, StructureFold is applicable to RNA structural profiling data obtained in vivo as well as to in vitro or in silico datasets. StructureFold is deployed via the Galaxy platform. AVAILABILITY AND IMPLEMENTATION: StructureFold is freely available as a component of Galaxy available at: https://usegalaxy.org/. CONTACT: yxt148@psu.edu or sma3@psu.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Algoritmos , Arabidopsis/genética , Genoma de Planta/genética , Conformação de Ácido Nucleico , RNA de Plantas/genética , RNA Ribossômico 18S/genética , RNA/química , Análise de Sequência de RNA/métodos , Biologia Computacional/métodos , Sequências Reguladoras de Ácido Ribonucleico/genética , TranscriptomaRESUMO
Guanine quadruplex structures (GQSs) play important roles in the regulation of gene expression and cellular processes. Recent studies provide strong evidence for the formation and function of DNA and RNA GQSs in human cells. However, whether GQSs form and are functional in plants remains essentially unexplored. On the basis of circular dichroism (CD)-detected titration, UV-detected melting, in-line probing (ILP) and reporter gene assay studies, we report the first example of a plant RNA GQS that inhibits translation. This GQS is located within the 5'-UTR of the ATAXIA TELANGIECTASIA-MUTATED AND RAD3-RELATED (ATR) mRNA of Arabidopsis thaliana (mouse-ear cress). We show that this GQS is highly stable and is thermodynamically favoured over a competing hairpin structure in the 5'-UTR at physiological K⺠and Mg²âº concentrations. Results from ILP reveal the secondary structure of the RNA and support formation of the GQS in vitro in the context of the complete 5'-UTR. Transient reporter gene assays performed in living plants reveal that the GQS inhibits translation but not transcription, implicating this GQS as a translational repressor in vivo. Our results provide the first complete demonstration of the formation and function of a regulatory RNA GQS in plants and open new avenues to explore potential functional roles of GQS in the plant kingdom.
Assuntos
Regiões 5' não Traduzidas , Arabidopsis/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quadruplex G , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Dicroísmo Circular , DNA Recombinante/química , DNA Recombinante/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica de Plantas , Genes Reporter , Sequências Repetidas Invertidas , Mutação , Desnaturação de Ácido Nucleico , Folhas de Planta/metabolismo , Dobramento de RNA , Estabilidade de RNA , RNA Mensageiro/química , RNA de Plantas/química , Especificidade da Espécie , Nicotiana/metabolismoRESUMO
The spike architecture of wheat plays a crucial role in determining grain number, making it a key trait for optimization in wheat breeding programs. In this study, we used a multi-omic approach to analyze the transcriptome and epigenome profiles of the young spike at eight developmental stages, revealing coordinated changes in chromatin accessibility and H3K27me3 abundance during the flowering transition. We constructed a core transcriptional regulatory network (TRN) that drives wheat spike formation and experimentally validated a multi-layer regulatory module involving TaSPL15, TaAGLG1, and TaFUL2. By integrating the TRN with genome-wide association studies, we identified 227 transcription factors, including 42 with known functions and 185 with unknown functions. Further investigation of 61 novel transcription factors using multiple homozygous mutant lines revealed 36 transcription factors that regulate spike architecture or flowering time, such as TaMYC2-A1, TaMYB30-A1, and TaWRKY37-A1. Of particular interest, TaMYB30-A1, downstream of and repressed by WFZP, was found to regulate fertile spikelet number. Notably, the excellent haplotype of TaMYB30-A1, which contains a C allele at the WFZP binding site, was enriched during wheat breeding improvement in China, leading to improved agronomic traits. Finally, we constructed a free and open access Wheat Spike Multi-Omic Database (http://39.98.48.156:8800/#/). Our study identifies novel and high-confidence regulators and offers an effective strategy for dissecting the genetic basis of wheat spike development, with practical value for wheat breeding.