NAD+-capped RNAs are preferentially produced from inducible genes in response to growth conditions in E. coli
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ABSTRACT: Recent findings regarding NAD+-capped RNAs (NAD-RNAs) indicate that prokaryotes and eukaryotes employ non-canonical RNA capping to regulate genes, a previously unrecognized mechanism. Two methods for transcriptome-wide analysis of NAD-RNAs, NAD captureSeq and NAD tagSeq, are based on copper-catalyzed azide-alkyne cycloaddition click chemistry reaction to label NAD-RNAs. However, copper can fragment RNA, interfering with the analyses. Here we report development of NAD tagSeq II, which uses copper-free, strain-promoted azide-alkyne cycloaddition for labeling NAD-RNAs, followed by identification of tagged RNA by direct RNA sequencing. Using this method, we compared NAD-RNA and total transcript profiles of E. coli cells in the exponential and stationary phases and identified hundreds of NAD-RNA species. For some genes, the majority of their transcripts were found as NAD-RNAs. Our study indicates that NAD-RNAs are preferentially produced from inducible genes in response to different growth conditions.
Project description:We developed a Copper-free, strain-promoted azide-alkyne cycloaddition reaction (SPAAC) to capture NAD-RNAs without RNA degradation. We examined the specificity of CuAAC and SPAAC reactions towards NAD+ vs. m7G, and found that both prefer NAD+ but also act on m7G. We show that m7G-capped RNA can be immuno-depleted, allowing for the specific identification of NAD-RNA via the SPAAC reaction and sequencing, which we name SPAAC-NAD-seq. Subjecting Arabidopsis RNA to both the original NAD captureSeq and SPAAC-NAD-seq, we found that more NAD+-capped RNA was identified by the latter, particularly those with low abundance. This led to the discovery of new gene ontology terms such as starch biosynthsis, intracellular protein transport and response to cadmium stress associated with genes that produce NAD-RNA. Furthermore, reads were uniformly distributed along gene bodies, which suggested that SPAAC-NAD-seq retained full-length sequence information. SPAAC-NAD-seq enables specific and efficient discovery of NAD-RNA in prokaryotes, and when combined with m7G-RNA depletion, in eukaryotes.
Project description:The 5’ end of a eukaryotic mRNA generally has a methyl guanosine cap (m7G cap) that not only protects the mRNA from degradation but also mediates almost all other aspects of gene expression. Some RNAs in E. coli, yeast, and mammals were recently found to contain an NAD+ cap at their 5’ ends. Here we report development of a new method – NAD tagSeq – for transcriptome-wide identification and quantification of NAD+-capped RNAs (NAD-RNAs). The method uses first an enzymatic reaction and then a click chemistry reaction to label NAD-RNAs with a synthetic RNA tag. The tagged RNA molecules can be enriched and directly sequenced using the Oxford Nanopore sequencing technology. NAD tagSeq not only allows more accurate identification and quantification of NAD-RNAs but can also reveal sequences of whole NAD-RNA transcripts. Using NAD tagSeq, we found that NAD-RNAs in Arabidopsis are mostly produced from a few thousand protein-coding genes, with over 60% of them from fewer than 200 genes. The top 2,000 genes that were found to produce the highest numbers of NAD-RNAs were enriched in the gene ontology terms of responses to oxidative stress and other stresses, photosynthesis, and protein synthesis. For some Arabidopsis genes, over 10% of their transcripts could be NAD-capped. The NAD-RNAs in Arabidopsis have similar overall sequence structures to their canonical m7G-capped mRNAs. The identification and quantification of NAD-RNAs and revealing their sequence features provide essential steps toward understanding functions of NAD-RNAs.
Project description:The method hijacks RNA methyltransferase activity to introduce an alkyne, instead of a methyl, moiety on RNA. Subsequent copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) with an imidazole-degrader leads to RNA cleavage and degradation, exploiting a mechanism used by endogenous ribonucleases. Focusing on N6-methyladenosine (m6A), we compare Slick-Seq to current state-of-the-art methods used to study of this modification, and show that the platform identifies methylated transcripts, determines RNA methylase specificity, and reliably maps modification sites in intronic and intergenic regions. Importantly, we discovered that METTL16 deposits m6A to intronic polyadenylation (IPA) sites, which suggests a potential role of METTL16 in IPA and, in turn, splicing. Unlike previously reported methods, Slick-Seq allows a comprehensive and dynamic study of RNA modifications throughout the transcriptome, including regions of low abundance.
Project description:Transcription is the primary regulatory step in gene expression coordinating the coding and non-coding genomic regions across space and time. Divergent initiation of transcription from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers. Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity from cell populations. However, the lack of single-cell resolved view of active transcription has left fundamental questions in gene regulation unanswered. In this study, we present scGRO-seq - a single-cell nascent RNA sequencing assay using copper-catalyzed azide-alkyne cycloaddition - unveiling the coordinated regulation of dynamic transcription throughout the genome. scGRO-seq demonstrates the episodic nature of transcription and provides estimates of burst size and frequency by directly quantifying transcribing RNA polymerases. It reveals the co-transcription of functionally related genes and leverages the transcription of replication-dependent non-polyadenylated histone genes to elucidate cell-cycle dynamics. The single-nucleotide spatiotemporal resolution of scGRO-seq characterizes networks of enhancers and genes and indicates the bursting of transcription at super-enhancers before the activation of burst from associated genes. Our method offers insights into the dynamic nature of transcription, functional architecture of the genome, and serves as a powerful tool to investigate the role of the non-coding genome in gene regulation.
Project description:Direct measurement of nucleosome turnover dynamics by using co-translational incorporation of the methionine (Met) surrogate azidohomoalaine (Aha) into proteins and subsequent ligation of biotin to Aha-containing proteins through the [3+2] cycloaddition reaction between the azide group of Aha and an alkyne linked to biotin. To measure turnover rates, we treat cells briefly with Aha, couple biotin to nucleosomes containing newly incorporated histones, affinity purify with strepavidin, wash stringently to remove non-histone proteins and H2A/H2B dimers, and analyze the affinity-purified DNA using tiling microarrays. We call this strategy 'CATCH-IT' for Covalent Attachment of Tags to Capture Histones and Identify Turnover. Keywords: Chromatin affinity-purification on microarray All experiments were done using strepavidin pulldown DNA cohybridized with total input DNA to the same array. Two channels per array, Cy5 and Cy3, were used in each experiment.
Project description:We design and test a novel di-azido LASER reagent capable enrichment through attachment of biotin with strain-promoted azide alkyne cycloaddition (SPAAC). We term this approach in vivo click LASER or icLASER. Aligned with the goal of extending transcriptome-wide measurements of RNA structure and to develop an approach that takes advantage of combinatorial RNA structure probing,we then use this novel bi-functional probe to interrogate LASER reactivity transcriptome-wide, revealing the first solvent accessibility transcriptome map. We also directly compare icSHAPE (hydroxyl acylation; flexibility) and icLASER (solvent accessibility) to demonstrate the power of utilizing them together to predict RNA-protein interactions and RNA polyadenylation.Our results demonstrate that combinatorial RNA structure probing can be employed to compliment orthogonal methods to better understand RNA structure and processing in cells transcriptome-wide.
Project description:The hub metabolite, nicotinamide adenine dinucleotide (NAD), can be used as an initiating nucleotide in RNA synthesis to result in NAD-capped RNAs (NAD-RNA). Since NAD has been heightened as one of the most essential modulators in aging and various age-related diseases, its attachment to RNA might indicate a yet-to-be discovered mechanism that impacts adult life-course. However, the unknown identity of NAD-linked RNAs in adult and aging tissues has hindered functional studies. Here, we introduce ONE-seq method to identify the RNA transcripts that contain NAD cap. ONE-seq has been optimized to use only one-step chemo-enzymatic biotinylation, followed by streptavidin capture and the nudix phosphohydrolase NudC-catalyzed elution, to specifically recover NAD-capped RNAs for epitranscriptome and gene-specific analyses. Our data describes more than a thousand of previously unknown NAD-RNAs in the mouse liver and reveals epitranscriptome-wide dynamics of NAD-RNAs with age.ONE-seq empowers the identification of NAD-capped RNAs that are responsive to distinct physiological states, facilitating functional investigation into this modification.
Project description:Direct measurement of nucleosome turnover dynamics by using co-translational incorporation of the methionine (Met) surrogate azidohomoalaine (Aha) into proteins and subsequent ligation of biotin to Aha-containing proteins through the [3+2] cycloaddition reaction between the azide group of Aha and an alkyne linked to biotin. To measure turnover rates, we treat cells briefly with Aha, couple biotin to nucleosomes containing newly incorporated histones, affinity purify with strepavidin, wash stringently to remove non-histone proteins and H2A/H2B dimers, and analyze the affinity-purified DNA using tiling microarrays. We call this strategy 'CATCH-IT' for Covalent Attachment of Tags to Capture Histones and Identify Turnover. Keywords: Chromatin affinity-purification on microarray
Project description:We developed a novel approach to in situ crosslinking mass spectrometry (XL-MS) where the reaction is split into two sequential and orthogonal coupling events. The method involves pre-stabilization of the proteome using a fixation protocol, followed by a two-step process that begins with extensive labeling of surface-available lysines with click-chemistry compatible reagents modified with NHS Esters. The second step involves the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction of the installed precursors, which generates crosslinks with high efficiency. We observed approximately a 20-fold increase in protein-protein interactions detected by this approach when compared to standard DSS crosslinkers, and our Click-linking strategy even outperformed enrichable crosslinkers such as PhoX.
Project description:17-Hydroxydocosahexaenoic acid (17-HDHA), an oxidative metabolite of the fish oil constituent docosahexaenoic acid (DHA, 22:6 n-3), is a signaling lipid with anti-inflammatory properties. The molecular mechanisms underlying the biological effect of 17-HDHA are poorly understood. Here, we report the design, synthesis and application of a complementary pair of bioorthogonal photoreactive probes based on the polyunsaturated scaffold of 17-HDHA and DHA. In these probes, an alkyne serves as a handle to introduce a fluorescent reporter group or a biotin-affinity tag via copper(I)-catalyzed azide-alkyne cycloaddition. This pair of chemical probes was used to map specific protein interaction partners of 17-HDHA in primary human macrophages, which led to the identification of prostaglandin reductase 1 (PTGR1) as a target of 17-HDHA, but not DHA. Ensuing biochemical studies revealed that PTGR1 converted 17-HDHA into 17-oxo-DHA, which inhibited the biosynthesis of the pro-inflammatory lipids 5-HETE and LTB4 in human macrophages and neutrophils.