Determination of RNA-ligand interactions with the photoaffinity platform PEARL-seq.

In the development of therapeutics, it is important to establish engagement of a compound to its intended target and identify other targets it binds to. Methods for demonstrating target engagement in the growing field of RNA-targeted therapeutics are therefore needed. We present a detailed protocol for Photoaffinity Evaluation of RNA Ligation-Sequencing (PEARL-seq), a platform for determining interactions between small molecule ligands and their target RNA(s).

NAD-seq for profiling the NAD capped transcriptome of .

Eukaryotic RNAs can be modified with a non-canonical 5' nicotinamide adenine dinucleotide (NAD) cap. NAD-seq identifies transcriptome-wide NAD capped RNAs. NAD-seq takes advantage of click chemistry to allow the capture of NAD capped RNAs.

Messenger RNA 5' NAD Capping Is a Dynamic Regulatory Epitranscriptome Mark That Is Required for Proper Response to Abscisic Acid in Arabidopsis.

Although eukaryotic messenger RNAs (mRNAs) normally possess a 5' end N-methyl guanosine (mG) cap, a non-canonical 5' nicotinamide adenine dinucleotide (NAD) cap can tag certain transcripts for degradation mediated by the NAD decapping enzyme DXO1. Despite this importance, whether NAD capping dynamically responds to specific stimuli to regulate eukaryotic transcriptomes remains unknown. Here, we reveal a link between NAD capping and tissue- and hormone response-specific mRNA stability.

PEARL-seq: A Photoaffinity Platform for the Analysis of Small Molecule-RNA Interactions.

RNA is emerging as a valuable target for the development of novel therapeutic agents. The rational design of RNA-targeting small molecules, however, has been hampered by the relative lack of methods for the analysis of small molecule-RNA interactions. Here, we present our efforts to develop such a platform using photoaffinity labeling.

HAMR: High-Throughput Annotation of Modified Ribonucleotides.

Ribonucleotides can be decorated with over 100 types of covalent chemical modifications. These modifications change the structure, function, and catalytic activity of RNAs, forming a layer of posttranscriptional regulation termed the epitranscriptome. Recent advances in high-throughput mapping have demonstrated these modifications are abundant and mark nearly all classes of RNAs, including messenger RNAs.

N-Methyladenosine Inhibits Local Ribonucleolytic Cleavage to Stabilize mRNAs in Arabidopsis.

N-methyladenosine (mA) is a dynamic, reversible, covalently modified ribonucleotide that occurs predominantly toward 3' ends of eukaryotic mRNAs and is essential for their proper function and regulation. In Arabidopsis thaliana, many RNAs contain at least one mA site, yet the transcriptome-wide function of mA remains mostly unknown. Here, we show that many mA-modified mRNAs in Arabidopsis have reduced abundance in the absence of this mark.

New insights into the plant epitranscriptome.

Throughout all kingdoms of life, ribonucleotides are marked with covalent chemical modifications that change the structure and binding properties of modified RNA molecules. These marks are deposited by 'writer' proteins, recognized by 'readers', and removed by 'erasers', thus forming an epitranscriptomic system of marks and binding proteins directly analogous to the epigenome. Recent advances in marrying classical biochemical techniques with high-throughput sequencing have enabled detailed mapping of plant epitranscriptomic marks, which in turn yielded insights into how these marks regulate a host of biological processes, from shoot stem cell fate to floral transition and from leaf development to viral activity.

Reading the Epitranscriptome: New Techniques and Perspectives.

Ribonucleotides can be covalently modified with over 100 known chemical moieties, greatly expanding the potential coding and regulatory repertoire of RNA. Recent advances in applying high-throughput sequencing to the study of RNA modifications have generated transcriptome-wide modification maps and demonstrated that modifications are abundant features of multiple classes of RNAs, including messenger RNAs. In turn, new regulatory functions for RNA modifications have been elucidated.

In Silico Identification of RNA Modifications from High-Throughput Sequencing Data Using HAMR.

RNA molecules are often altered post-transcriptionally by the covalent modification of their nucleotides. These modifications are known to modulate the structure, function, and activity of RNAs. When reverse transcribed into cDNA during RNA sequencing library preparation, atypical (modified) ribonucleotides that affect Watson-Crick base pairing will interfere with reverse transcriptase (RT), resulting in cDNA products with mis-incorporated bases or prematurely terminated RNA products.

The Conservation and Function of RNA Secondary Structure in Plants.

RNA transcripts fold into secondary structures via intricate patterns of base pairing. These secondary structures impart catalytic, ligand binding, and scaffolding functions to a wide array of RNAs, forming a critical node of biological regulation. Among their many functions, RNA structural elements modulate epigenetic marks, alter mRNA stability and translation, regulate alternative splicing, transduce signals, and scaffold large macromolecular complexes.

Chemical Modifications Mark Alternatively Spliced and Uncapped Messenger RNAs in Arabidopsis.

Posttranscriptional chemical modification of RNA bases is a widespread and physiologically relevant regulator of RNA maturation, stability, and function. While modifications are best characterized in short, noncoding RNAs such as tRNAs, growing evidence indicates that mRNAs and long noncoding RNAs (lncRNAs) are likewise modified. Here, we apply our high-throughput annotation of modified ribonucleotides (HAMR) pipeline to identify and classify modifications that affect Watson-Crick base pairing at three different levels of the Arabidopsis thaliana transcriptome (polyadenylated, small, and degrading RNAs).

Transcriptome-wide measurement of plant RNA secondary structure.

RNAs fold into intricate and precise secondary structures. These structural patterns regulate multiple steps of the RNA lifecycle, while also conferring catalytic and scaffolding functions to certain transcripts. Therefore, a full understanding of RNA posttranscriptional regulation requires a comprehensive picture of secondary structure.

High-throughput nuclease-mediated probing of RNA secondary structure in plant transcriptomes.

Empirical measurement of RNA secondary structure is an invaluable tool that has provided a more complete understanding of the RNA life cycle and functionality of this extremely important molecule. In general, methods for probing structural information involve treating RNA with either a chemical or an enzyme that preferentially targets regions of the RNA in a single- or double-stranded conformation (ssRNA and dsRNA, respectively). Here, we describe an approach that utilizes a combination of ssRNA- and dsRNA-specific nuclease (ss- and dsRNase, respectively) treatments along with high-throughput sequencing technology to provide comprehensive and robust measurements of RNA secondary structure across entire plant transcriptomes.

Essential Role for endogenous siRNAs during meiosis in mouse oocytes.

The RNase III enzyme DICER generates both microRNAs (miRNAs) and endogenous short interfering RNAs (endo-siRNAs). Both small RNA species silence gene expression post-transcriptionally in association with the ARGONAUTE (AGO) family of proteins. In mammals, there are four AGO proteins (AGO1-4), of which only AGO2 possesses endonucleolytic activity.

Detection of Pol IV/RDR2-dependent transcripts at the genomic scale in Arabidopsis reveals features and regulation of siRNA biogenesis.

Twenty-four-nucleotide small interfering (si)RNAs are central players in RNA-directed DNA methylation (RdDM), a process that establishes and maintains DNA methylation at transposable elements to ensure genome stability in plants. The plant-specific RNA polymerase IV (Pol IV) is required for siRNA biogenesis and is believed to transcribe RdDM loci to produce primary transcripts that are converted to double-stranded RNAs (dsRNAs) by RDR2 to serve as siRNA precursors. Yet, no such siRNA precursor transcripts have ever been reported.

Regulatory impact of RNA secondary structure across the Arabidopsis transcriptome.

The secondary structure of an RNA molecule plays an integral role in its maturation, regulation, and function. However, the global influence of this feature on plant gene expression is still largely unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach in conjunction with transcriptome-wide sequencing of rRNA-depleted (RNA sequencing), small RNA, and ribosome-bound RNA populations to investigate the impact of RNA secondary structure on gene expression regulation in Arabidopsis thaliana.

Succession in the gut microbiome following antibiotic and antibody therapies for Clostridium difficile.

Antibiotic disruption of the intestinal microbiota may cause susceptibility to pathogens that is resolved by progressive bacterial outgrowth and colonization. Succession is central to ecological theory but not widely documented in studies of the vertebrate microbiome. Here, we study succession in the hamster gut after treatment with antibiotics and exposure to Clostridium difficile.