Over 100 distinct chemical modifications can be catalyzed on RNA post-synthesis, potentially serving as a post-transcriptional regulatory layer of gene expression. This review focuses on recent ...advances, knowledge gaps, and challenges pertaining to N6-methyladenosine (m6A), an abundant modification of mRNA for which substantial progress has been made in recent years. The discussed aspects are also very relevant for a wide range of additional modifications on mRNA collectively coined the epitranscriptome.
Circular RNA forms had been described in all domains of life. Such RNAs were shown to have diverse biological functions, including roles in the life cycle of viral and viroid genomes, and in ...maturation of permuted tRNA genes. Despite their potentially important biological roles, discovery of circular RNAs has so far been mostly serendipitous. We have developed circRNA-seq, a combined experimental/computational approach that enriches for circular RNAs and allows profiling their prevalence in a whole-genome, unbiased manner. Application of this approach to the archaeon Sulfolobus solfataricus P2 revealed multiple circular transcripts, a subset of which was further validated independently. The identified circular RNAs included expected forms, such as excised tRNA introns and rRNA processing intermediates, but were also enriched with non-coding RNAs, including C/D box RNAs and RNase P, as well as circular RNAs of unknown function. Many of the identified circles were conserved in Sulfolobus acidocaldarius, further supporting their functional significance. Our results suggest that circular RNAs, and particularly circular non-coding RNAs, are more prevalent in archaea than previously recognized, and might have yet unidentified biological roles. Our study establishes a specific and sensitive approach for identification of circular RNAs using RNA-seq, and can readily be applied to other organisms.
An increasing body of evidence indicates that transcription and splicing are coupled, and it is accepted that chromatin organization regulates transcription. Little is known about the cross-talk ...between chromatin structure and exon-intron architecture. By analysis of genome-wide nucleosome-positioning data sets from humans, flies and worms, we found that exons show increased nucleosome-occupancy levels with respect to introns, a finding that we link to differential GC content and nucleosome-disfavoring elements between exons and introns. Analysis of genome-wide chromatin immunoprecipitation data in humans and mice revealed four specific post-translational histone modifications enriched in exons. Our findings indicate that previously described enrichment of H3K36me3 modifications in exons reflects a more fundamental phenomenon, namely increased nucleosome occupancy along exons. Our results suggest an RNA polymerase II-mediated cross-talk between chromatin structure and exon-intron architecture, implying that exon selection may be modulated by chromatin structure.
Nascent messenger RNA is endowed with a poly(A) tail that is subject to gradual deadenylation and subsequent degradation in the cytoplasm. Deadenylation and degradation rates are typically ...correlated, rendering it difficult to dissect the determinants governing each of these processes and the mechanistic basis of their coupling. Here we developed an approach that allows systematic, robust and multiplexed quantification of poly(A) tails in Saccharomyces cerevisiae. Our results suggest that mRNA deadenylation and degradation rates are decoupled during meiosis, and that transcript length is a major determinant of deadenylation rates and a key contributor to reshaping of poly(A) tail lengths. Meiosis-specific decoupling also leads to unique positive associations between poly(A) tail length and gene expression. The decoupling is associated with a focal localization pattern of the RNA degradation factor Xrn1, and can be phenocopied by Xrn1 deletion under nonmeiotic conditions. Importantly, the association of transcript length with deadenylation rates is conserved across eukaryotes. Our study uncovers a factor that shapes deadenylation rate and reveals a unique context in which degradation is decoupled from deadenylation.
N6-methyladenosine (m6A) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify ...m6A stoichiometry and the lack of antibody-independent methodologies for interrogating m6A. Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%–25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of m6A sites, calibration of the performance of antibody-based approaches, and quantitative tracking of m6A dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is “hard coded” in cis via a simple and predictable code, accounting for 33%–46% of the variability in methylation levels and allowing accurate prediction of m6A loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of m6A regulation in subcellular fractions, diverse cell types, and disease states.
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•RNA digestion via m6A sensitive RNase (MAZTER-seq) allows systematic m6A quantitation•MAZTER-seq reveals that antibody-based methods are of limited sensitivity•m6A stoichiometry is “hard coded” by a simple, predictable, and conserved code•MAZTER-seq allows quantitative tracking of m6A in diverse biological settings
A new enzymatic approach for precise mapping and measurement of m6A within mRNAs provides insight into how methylation sites are selected and the functional impact of the modifications.
Modifications on mRNA offer the potential of regulating mRNA fate post-transcriptionally. Recent studies suggested the widespread presence of N
-methyladenosine (m
A), which disrupts Watson-Crick ...base pairing, at internal sites of mRNAs. These studies lacked the resolution of identifying individual modified bases, and did not identify specific sequence motifs undergoing the modification or an enzymatic machinery catalysing them, rendering it challenging to validate and functionally characterize putative sites. Here we develop an approach that allows the transcriptome-wide mapping of m
A at single-nucleotide resolution. Within the cytosol, m
A is present in a low number of mRNAs, typically at low stoichiometries, and almost invariably in tRNA T-loop-like structures, where it is introduced by the TRMT6/TRMT61A complex. We identify a single m
A site in the mitochondrial ND5 mRNA, catalysed by TRMT10C, with methylation levels that are highly tissue specific and tightly developmentally controlled. m
A leads to translational repression, probably through a mechanism involving ribosomal scanning or translation. Our findings suggest that m
A on mRNA, probably because of its disruptive impact on base pairing, leads to translational repression, and is generally avoided by cells, while revealing one case in mitochondria where tight spatiotemporal control over m
A levels was adopted as a potential means of post-transcriptional regulation.
How are short exonic sequences recognized within the vast intronic oceans in which they reside? Despite decades of research, this remains one of the most fundamental, yet enigmatic, questions in the ...field of pre‐mRNA splicing research. For many years, studies aiming to shed light on this process were focused at the RNA level, characterizing the manner by which splicing factors and auxiliary proteins interact with splicing signals, thereby enabling, facilitating and regulating splicing. However, we increasingly understand that splicing is not an isolated process; rather it occurs co‐transcriptionally and is presumably also regulated by transcription‐related processes. In fact, studies by our group and others over the past year suggest that DNA structure in terms of nucleosome positioning and specific histone modifications, which have a well established role in transcription, may also have a role in splicing. In this review we discuss evidence for the coupling between transcription and splicing, focusing on recent findings suggesting a link between chromatin structure and splicing, and highlighting challenges this emerging field is facing.
Following synthesis, RNA can be modified with over 100 chemically distinct modifications, which can potentially regulate RNA expression post-transcriptionally. Pseudouridine (Ψ) was recently ...established to be widespread and dynamically regulated on yeast mRNA, but less is known about Ψ presence, regulation, and biogenesis in mammalian mRNA. Here, we sought to characterize the Ψ landscape on mammalian mRNA, to identify the main Ψ-synthases (PUSs) catalyzing Ψ formation, and to understand the factors governing their specificity toward selected targets. We first developed a framework allowing analysis, evaluation, and integration of Ψ mappings, which we applied to >2.5 billion reads from 30 human samples. These maps, complemented with genetic perturbations, allowed us to uncover TRUB1 and PUS7 as the two key PUSs acting on mammalian mRNA and to computationally model the sequence and structural elements governing the specificity of TRUB1, achieving near-perfect prediction of its substrates (AUC = 0.974). We then validated and extended these maps and the inferred specificity of TRUB1 using massively parallel reporter assays in which we monitored Ψ levels at thousands of synthetically designed sequence variants comprising either the sequences surrounding pseudouridylation targets or systematically designed mutants perturbing RNA sequence and structure. Our findings provide an extensive and high-quality characterization of the transcriptome-wide distribution of pseudouridine in human and the factors governing it and provide an important resource for the community, paving the path toward functional and mechanistic dissection of this emerging layer of post-transcriptional regulation.
A prerequisite to defining the transcriptome-wide functions of RNA modifications is the ability to accurately determine their location. Here, we present N4-acetylcytidine (ac4C) sequencing ...(ac4C-seq), a protocol for the quantitative single-nucleotide resolution mapping of cytidine acetylation in RNA. This method exploits the kinetically facile chemical reaction of ac4C with sodium cyanoborohydride under acidic conditions to form a reduced nucleobase. RNA is then fragmented, ligated to an adapter at its 3' end and reverse transcribed to introduce a non-cognate nucleotide at reduced ac4C sites. After adapter ligation, library preparation and high-throughput sequencing, a bioinformatic pipeline enables identification of ac4C positions on the basis of the presence of C→T misincorporations in reduced samples but not in controls. Unlike antibody-based approaches, ac4C-seq identifies specific ac4C residues and reports on their level of modification. The ac4C-seq library preparation protocol can be completed in ~4 d for transcriptome-wide sequencing.
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