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). PEARL-seq allows detection of binding and crosslinking events with single nucleotide resolution and allows measurement of enrichment of the target RNA relative to all other RNAs. PEARL-seq is a valuable tool in the effort to verify bona fide RNA-ligand interactions.

Plant 24-nt reproductive phasiRNAs from intramolecular duplex mRNAs in diverse monocots.

In grasses, two pathways that generate diverse and numerous 21-nt (premeiotic) and 24-nt (meiotic) phased siRNAs are highly enriched in anthers, the male reproductive organs. These "phasiRNAs" are analogous to mammalian piRNAs, yet their functions and evolutionary origins remain largely unknown. The 24-nt meiotic phasiRNAs have only been described in grasses, wherein their biogenesis is dependent on a specialized Dicer (DCL5). To assess how evolution gave rise to this pathway, we examined reproductive phasiRNA pathways in nongrass monocots: garden asparagus, daylily, and lily. The common ancestors of these species diverged approximately 115-117 million years ago (MYA). We found that premeiotic 21-nt and meiotic 24-nt phasiRNAs were abundant in all three species and displayed spatial localization and temporal dynamics similar to grasses. The miR2275-triggered pathway was also present, yielding 24-nt reproductive phasiRNAs, and thus originated more than 117 MYA. In asparagus, unlike in grasses, these siRNAs are largely derived from inverted repeats (IRs); analyses in lily identified thousands of precursor loci, and many were also predicted to form foldback substrates for Dicer processing. Additionally, reproductive phasiRNAs were present in female reproductive organs and thus may function in both male and female germinal development. These data describe several distinct mechanisms of production for 24-nt meiotic phasiRNAs and provide new insights into the evolution of reproductive phasiRNA pathways in monocots.

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. Here, through genome-wide profiling of RNAs in genotypes that compromise the processing of siRNA precursors, we were able to identify Pol IV/RDR2-dependent transcripts from tens of thousands of loci. We show that Pol IV/RDR2-dependent transcripts correspond to both DNA strands, whereas the RNA polymerase II (Pol II)-dependent transcripts produced upon derepression of the loci are derived primarily from one strand. We also show that Pol IV/RDR2-dependent transcripts have a 5' monophosphate, lack a poly(A) tail at the 3' end, and contain no introns; these features distinguish them from Pol II-dependent transcripts. Like Pol II-transcribed genic regions, Pol IV-transcribed regions are flanked by A/T-rich sequences depleted in nucleosomes, which highlights similarities in Pol II- and Pol IV-mediated transcription. Computational analysis of siRNA abundance from various mutants reveals differences in the regulation of siRNA biogenesis at two types of loci that undergo CHH methylation via two different DNA methyltransferases. These findings begin to reveal features of Pol IV/RDR2-mediated transcription at the heart of genome stability in plants.

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. In this review, we highlight recent developments in the study of plant epitranscriptomics, with an emphasis on N6-methyladenosine (m6A) and 5-methylcytosine (m5C). These studies have advanced the field beyond descriptive mapping or isolated genetic studies, and produced a more nuanced understanding of how components of the epitranscriptome and their binding proteins directly regulate critical aspects of plant biology.

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). We find this type of modifications primarily within uncapped, degrading mRNAs and lncRNAs, suggesting they are the cause or consequence of RNA turnover. Additionally, modifications within stable mRNAs tend to occur in alternatively spliced introns, suggesting they regulate splicing. Furthermore, these modifications target mRNAs with coherent functions, including stress responses. Thus, our comprehensive analysis across multiple RNA classes yields insights into the functions of covalent RNA modifications in 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. siRNAs trigger endonucleolytic cleavage of target mRNAs, mediated by AGO2, whereas miRNAs cause translational repression and mRNA decay through association with any of the four AGO proteins. Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because mouse oocytes express both miRNAs and endo-siRNAs, this phenotype could be due to the absence of either class of small RNA, or both. However, we and others demonstrated that miRNA function is suppressed in mouse oocytes, which suggested that endo-siRNAs, not miRNAs, are essential for female meiosis. To determine if this was the case we generated mice that express a catalytically inactive knock-in allele of Ago2 (Ago2ADH) exclusively in oocytes and thereby disrupted the function of siRNAs. Oogenesis and hormonal response are normal in Ago2ADH oocytes, but meiotic maturation is impaired, with severe defects in spindle formation and chromosome alignment that lead to meiotic catastrophe. The transcriptome of these oocytes is widely perturbed and shows a highly significant correlation with the transcriptome of Dicer null and Ago2 null oocytes. Expression of the mouse transcript (MT), the most abundant transposable element in mouse oocytes, is increased. This study reveals that endo-siRNAs are essential during meiosis I in mouse females, demonstrating a role for endo-siRNAs in mammals.

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. From this analysis, we find that highly unpaired and paired RNAs are strongly correlated with euchromatic and heterochromatic epigenetic histone modifications, respectively, providing evidence that secondary structure is necessary for these RNA-mediated posttranscriptional regulatory pathways. Additionally, we uncover key structural patterns across protein-coding transcripts that indicate RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in this model plant. We further reveal that RNA folding is significantly anticorrelated with overall transcript abundance, which is often due to the increased propensity of highly structured mRNAs to be degraded and/or processed into small RNAs. Finally, we find that secondary structure affects mRNA translation, suggesting that this feature regulates plant gene expression at multiple levels. These findings provide a global assessment of RNA folding and its significant regulatory effects in a plant transcriptome.

NAD-seq for profiling the NAD+ capped transcriptome of Arabidopsis thaliana.

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. Unlike other approaches, NAD-seq does not require DNA synthesis on beads, but this technique uses full NAD+ capped transcripts eluted from beads as the substrates for strand-specific RNA sequencing library preparation. For complete details on the use and execution of this protocol, please refer to Yu et al. (2021).

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 N7-methyl guanosine (m7G) 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. In the absence of DXO1 function, transcripts displaying a high proportion of NAD+ capping are instead processed into RNA-dependent RNA polymerase 6-dependent small RNAs, resulting in their continued turnover likely to free the NAD+ molecules. Additionally, the NAD+-capped transcriptome is significantly remodeled in response to the essential plant hormone abscisic acid in a mechanism that is primarily independent of DXO1. Overall, our findings reveal a previously uncharacterized and essential role of NAD+ capping in dynamically regulating transcript stability during specific physiological responses.

N6-methyladenosine and RNA secondary structure affect transcript stability and protein abundance during systemic salt stress in Arabidopsis.

After transcription, a messenger RNA (mRNA) is further post-transcriptionally regulated by several features including RNA secondary structure and covalent RNA modifications (specifically N6-methyladenosine, m6A). Both RNA secondary structure and m6A have been demonstrated to regulate mRNA stability and translation and have been independently linked to plant responses to soil salinity levels. However, the effect of m6A on regulating RNA secondary structure and the combinatorial interplay between these two RNA features during salt stress response has yet to be studied. Here, we globally identify RNA-protein interactions and RNA secondary structure during systemic salt stress. This analysis reveals that RNA secondary structure changes significantly during salt stress, and that it is independent of global changes in RNA-protein interactions. Conversely, we find that m6A is anti-correlated with RNA secondary structure in a condition-dependent manner, with salt-specific m6A correlated with a decrease in mRNA secondary structure during salt stress. Taken together, we suggest that salt-specific m6A deposition and the associated loss of RNA secondary structure results in increases in mRNA stability for transcripts encoding abiotic stress response proteins and ultimately increases in protein levels from these stabilized transcripts. In total, our comprehensive analyses reveal important post-transcriptional regulatory mechanisms involved in plant long-term salt stress response and adaptation.

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. This technique, termed Photoaffinity Evaluation of RNA Ligation-Sequencing (PEARL-seq), enables the rapid identification of small molecule binding locations within their RNA targets and can provide information on ligand selectivity across multiple different RNAs. These data, when supplemented with small molecule SAR data and RNA probing data enable the construction of a computational model of the RNA-ligand structure, thereby enabling the rational design of novel RNA-targeted ligands.

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. Here, we outline one such technique called high-throughput annotation of modified ribonucleotides (HAMR). HAMR exploits the tendency of certain modified ribonucleotides to interfere with base pairing, leading to errors in complementary DNA synthesis during RNA sequencing library preparation. In total, we present a computational protocol for in silico identification of modifications with HAMR, which can be retroactively applied to a variety of RNA sequencing techniques.

N6-Methyladenosine Inhibits Local Ribonucleolytic Cleavage to Stabilize mRNAs in Arabidopsis.

N6-methyladenosine (m6A) 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 m6A site, yet the transcriptome-wide function of m6A remains mostly unknown. Here, we show that many m6A-modified mRNAs in Arabidopsis have reduced abundance in the absence of this mark. The decrease in abundance is due to transcript destabilization caused by cleavage occurring 4 or 5 nt directly upstream of unmodified m6A sites. Importantly, we also find that, upon agriculturally relevant salt treatment, m6A is dynamically deposited on and stabilizes transcripts encoding proteins required for salt and osmotic stress response. Overall, our findings reveal that m6A generally acts as a stabilizing mark through inhibition of site-specific cleavage in plant transcriptomes, and this mechanism is required for proper regulation of the salt-stress-responsive transcriptome.

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. Here, we review both targeted and global methods for surveying RNA modification, with a focus on how transcriptome-wide methods have helped expand the understanding of modification-mediated regulation of the transcriptome.

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. These interactions with RT can therefore be inferred from mismatch patterns in the sequencing reads, and are distinguishable from simple base-calling errors, single-nucleotide polymorphisms (SNPs), or RNA editing sites. Here, we describe a computational protocol for the in silico identification of modified ribonucleotides from RT-based RNA-seq read-out using the High-throughput Analysis of Modified Ribonucleotides (HAMR) software. HAMR can identify these modifications transcriptome-wide with single nucleotide resolution, and also differentiate between different types of modifications to predict modification identity. Researchers can use HAMR to identify and characterize RNA modifications using RNA-seq data from a variety of common RT-based sequencing protocols such as Poly(A), total RNA-seq, and small RNA-seq.

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. Thus, the study of RNA secondary structure is critical to understanding the function and regulation of RNA transcripts. Here, we review the origins, form, and function of RNA secondary structure, focusing on plants. We then provide an overview of methods for probing secondary structure, from physical methods such as X-ray crystallography and nuclear magnetic resonance (NMR) imaging to chemical and nuclease probing methods. Combining these latter methods with high-throughput sequencing has enabled them to scale across whole transcriptomes, yielding tremendous new insights into the form and function of RNA 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.

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. Here, we review several high throughput sequencing-based methods to globally survey plant RNA secondary structure. These methods are more accurate than computational prediction, and more scalable than physical techniques such as crystallography. We note hurdles to reliably measuring secondary structure, including RNA-binding proteins, RNA base modifications, and intramolecular duplexes. Finally, we survey the functional knowledge that has been gleaned from each of these methods, and identify some unanswered questions that remain.

The Msi Family of RNA-Binding Proteins Function Redundantly as Intestinal Oncoproteins.

Members of the Msi family of RNA-binding proteins have recently emerged as potent oncoproteins in a range of malignancies. MSI2 is highly expressed in hematopoietic cancers, where it is required for disease maintenance. In contrast to the hematopoietic system, colorectal cancers can express both Msi family members, MSI1 and MSI2. Here, we demonstrate that, in the intestinal epithelium, Msi1 and Msi2 have analogous oncogenic effects. Further, comparison of Msi1/2-induced gene expression programs and transcriptome-wide analyses of Msi1/2-RNA-binding targets reveal significant functional overlap, including induction of the PDK-Akt-mTORC1 axis. Ultimately, we demonstrate that concomitant loss of function of both MSI family members is sufficient to abrogate the growth of human colorectal cancer cells, and Msi gene deletion inhibits tumorigenesis in several mouse models of intestinal cancer. Our findings demonstrate that MSI1 and MSI2 act as functionally redundant oncoproteins required for the ontogeny of intestinal cancers.

Arabidopsis mRNA secondary structure correlates with protein function and domains.

RNAs fold into intricate structures that are determined by specific base pairing interactions encoded within their primary sequences. Recently, a number of transcriptome-wide studies have suggested that RNA secondary structure is a potent cis-acting regulator of numerous post-transcriptional processes in viruses and eukaryotes. However, the need for experimentally-based structure determination methods has not been well addressed. Here, we show that the regulatory significance of Arabidopsis RNA secondary structure is revealed specifically through high-throughput, sequencing-based, structure mapping data, not by computational prediction. Additionally, we find that transcripts with similar levels of secondary structure in their UTRs (5' or 3') or CDS tend to encode proteins with coherent functions. Finally, we reveal that portions of mRNAs encoding predicted protein domains are significantly more structured than those specifying inter-domain regions. In total, our findings show the utility of high-throughput, sequencing-based, structure-mapping approaches and suggest that mRNA folding regulates protein maturation and function.