Yiliang Ding

In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features

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.

Positioning the nodule, the hormone dictum

The formation of a nitrogen-fixing nodule involves two diverse developmental processes in the legume root: infection thread initiation in epidermal cells and nodule primordia formation in the cortex. Several plant hormones have been reported to positively or negatively regulate nodulation. These hormones function at different stages in the nodulation process and may facilitate the coordinated development of the epidermal and cortical developmental programmes that are necessary to allow bacterial infection into the developing nodule. In this paper, we review and discuss how the tissue specific nature of hormonal action dictates where, when and how a nodule is formed.

Determination of in vivo RNA structure in low-abundance transcripts

RNA structure plays important roles in diverse biological processes. However, the structures of all but the few most abundant RNAs are presently unknown in vivo. Here we introduce DMS/SHAPE-LMPCR to query the in vivo structures of low-abundance transcripts. DMS/SHAPE-LMPCR achieves attomole sensitivity, a 100,000-fold improvement over conventional methods. We probe the structure of low-abundance U12 small nuclear RNA (snRNA) in Arabidopsis thaliana and provide in vivo evidence supporting our derived phylogenetic structure. Interestingly, in contrast to mammalian U12 snRNAs, the loop of the SLIIb in U12 snRNA is variable among plant species, and DMS/SHAPE-LMPCR determines it to be unstructured. We reveal the effects of proteins on 25S rRNA, 5.8S rRNA and U12 snRNA structure, illustrating the critical importance of mapping RNA structure in vivo. Our universally applicable method opens the door to identifying and exploring the specific structure-function relationships of the multitude of low-abundance RNAs that prevail in living cells.

The NIN Transcription Factor Coordinates Diverse Nodulation Programs in Different Tissues of the Medicago truncatula Root

The Medicago truncatula transcription factor NIN coordinates diverse processes during nodulation, restricting ENOD11 expression in the root epidermis and promoting CRE1 expression in the root cortex. 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.

Genome-wide profiling of in vivo RNA structure at single-nucleotide resolution using structure-seq

Structure-seq is a high-throughput and quantitative method that provides genome-wide information on RNA structure at single-nucleotide resolution. Structure-seq can be performed both in vivo and in vitro to study RNA structure-function relationships, RNA regulation of gene expression and RNA processing. Structure-seq can be carried out by an experienced molecular biologist with a basic understanding of bioinformatics. Structure-seq begins with chemical RNA structure probing under single-hit kinetics conditions. Certain chemical modifications, e.g., methylation of the Watson-Crick face of unpaired adenine and cytosine residues by dimethyl sulfate, result in a stop in reverse transcription. Modified RNA is then subjected to reverse transcription using random hexamer primers, which minimizes 3′ end bias; reverse transcription proceeds until it is blocked by a chemically modified residue. Resultant cDNAs are amplified by adapter-based PCR and subjected to high-throughput sequencing, subsequently allowing retrieval of the structural information on a genome-wide scale. In contrast to classical methods that provide information only on individual transcripts, a single structure-seq experiment provides information on tens of thousands of RNA structures in ∼1 month. Although the procedure described here is for Arabidopsis thaliana seedlings in vivo, structure-seq is widely applicable, thereby opening new avenues to explore RNA structure–function relationships in living organisms.

StructureFold: genome-wide RNA secondary structure mapping and reconstruction in vivo

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.

A hybridization-based approach for quantitative and low-bias single-stranded DNA ligation.

Single-stranded DNA (ssDNA) ligation is a crucial step in many biochemical assays. Efficient ways of carrying out this reaction, however, are lacking. We show here that existing ssDNA ligation methods suffer from slow kinetics, poor yield, and severe nucleotide preference. To resolve these issues, we introduce a hybridization-based strategy that provides efficient and low-bias ligation of ssDNA. Our method uses a hairpin DNA to hybridize to any incoming acceptor ssDNA with low bias, with ligation of these strands mediated by T4 DNA ligase. This technique potentially can be applied in protocols that require ligation of ssDNA, including ligation-mediated polymerase chain reaction (LMPCR) and complementary DNA (cDNA) library construction.

Rice In Vivo RNA Structurome Reveals RNA Secondary Structure Conservation and Divergence in Plants

RNA secondary structure plays a critical role in gene regulation. Rice (Oryza 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 N6-methyladenosine (m6A) 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.

FoldAtlas: a repository for genome-wide RNA structure probing data.

Summary: 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.uk. Supplementary information: Supplementary data are available at Bioinformatics online.

Copper Ion Elicits Defense Response in Arabidopsis thaliana by Activating Salicylate- and Ethylene-Dependent Signaling Pathways

Copper, one of the micronutrients, is essential during vegetative growth, development, and multiplication of plants (Penarrubia et al., 2010). Copper is widely known for its application in fungicides and bactericides in agriculture since the late 19th century, for example, in the well-known Bordeaux mixture. Previous studies show that the mechanisms of copper ions-mediated antimicrobial activity involve either inhibition of the enzymes participated in fungal spore germination or direct toxicity on the growth of bacteria.