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LC-MS/MS analysis of human cellular tRNA

ABSTRACT: The field of epitranscriptomics is growing in importance, with chemical modification of RNA being associated with a wide variety of biological phenomena. Mass spectrometry (MS) enables the identification of modified RNA residues within their sequence contexts, by using analogous approaches to shotgun proteomics. We have developed a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), as part of the OpenMS software framework. NASE allows the reliable identification of (modified) RNA sequences from LC-MS/MS data in a high-throughput fashion. For this validation dataset, we generated samples of human total tRNA from a cellular extract - a complex mixture of highly modified RNAs. The samples were RNase-treated prior to nanoflow LC-MS/MS analysis.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Hendrik Weisser

LAB HEAD: Byron Andrews

PROVIDER: PXD016328 | Pride | 2020-03-31

REPOSITORIES: Pride

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A computational platform for high-throughput analysis of RNA sequences and modifications by mass spectrometry.

Wein Samuel S Andrews Byron B Sachsenberg Timo T Santos-Rosa Helena H Kohlbacher Oliver O Kouzarides Tony T Garcia Benjamin A BA Weisser Hendrik H

Nature communications 20200217 1

The field of epitranscriptomics continues to reveal how post-transcriptional modification of RNA affects a wide variety of biological phenomena. A pivotal challenge in this area is the identification of modified RNA residues within their sequence contexts. Mass spectrometry (MS) offers a comprehensive solution by using analogous approaches to shotgun proteomics. However, software support for the analysis of RNA MS data is inadequate at present and does not allow high-throughput processing. Exist ...[more]

PMID: 32066737

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LC-MS/MS analysis of human cellular tRNA

Project description:The field of epitranscriptomics is growing in importance, with chemical modification of RNA being associated with a wide variety of biological phenomena. Mass spectrometry (MS) enables the identification of modified RNA residues within their sequence contexts, by using analogous approaches to shotgun proteomics. We have developed a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), as part of the OpenMS software framework. NASE allows the reliable identification of (modified) RNA sequences from LC-MS/MS data in a high-throughput fashion. For this validation dataset, we generated a sample of human total tRNA from a cellular extract - a complex mixture of highly modified RNAs. This sample was RNase-treated prior to nanoflow LC-MS/MS analysis.

2018-12-31 | PXD012097 | Pride

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2020-03-31 | PXD016323 | Pride

MS-based analysis of enzymatic methylation of an in vitro-transcribed lncRNA

Project description:The field of epitranscriptomics is growing in importance, with chemical modification of RNA being associated with a wide variety of biological phenomena. Mass spectrometry (MS) enables the identification of modified RNA residues within their sequence contexts, by using analogous approaches to shotgun proteomics. We have developed a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), as part of the OpenMS software framework. NASE allows the reliable identification of (modified) RNA sequences from LC-MS/MS data in a high-throughput fashion. For this validation dataset, we prepared two samples of an in vitro-transcribed yeast lncRNA (NME1, 340 nt long), one of which was treated with an RNA methyltransferase (NCL1) catalyzing the 5-methylcytidine (m5C) modification. These samples were subsequently digested with an RNA endonuclease (RNase) to generate oligonucleotide sequences of a length amenable to mass spectrometry.

2018-12-31 | PXD012095 | Pride

MS-based analysis of enzymatic methylation of an in vitro-transcribed lncRNA

Project description:The field of epitranscriptomics is growing in importance, with chemical modification of RNA being associated with a wide variety of biological phenomena. Mass spectrometry (MS) enables the identification of modified RNA residues within their sequence contexts, by using analogous approaches to shotgun proteomics. We have developed a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), as part of the OpenMS software framework. NASE allows the reliable identification of (modified) RNA sequences from LC-MS/MS data in a high-throughput fashion. For this validation dataset, we prepared two samples of an in vitro-transcribed yeast lncRNA (NME1, 340 nt long), one of which was treated with an RNA methyltransferase (NCL1) catalyzing the 5-methylcytidine (m5C) modification. These samples were subsequently digested with an RNA endonuclease (RNase) to generate oligonucleotide sequences of a length amenable to mass spectrometry.

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LC-MS/MS analysis of an intact synthetic microRNA

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2019-01-10 | PXD012094 | Pride

Profiling 4-thiouridine across E. coli tRNAs

Project description:Profiling 4-thiouridine modification present at position 8 of E. coli transfer RNAs

2022-01-30 | PXD028405 | Pride

Human RNase 4 Improves mRNA Sequence Characterization by LC-MS/MS.

Project description:With the rapid growth of synthetic messenger RNA (mRNA)-based therapeutics and vaccines, the development of analytical tools for characterization of long, complex RNAs has become essential. Tandem liquid chromatography-mass spectrometry (LC-MS/MS) permits direct assessment of the mRNA primary sequence and modifications thereof without conversion to cDNA or amplification. It relies upon digestion of mRNA with site-specific endoribonucleases to generate pools of short oligonucleotides that are then amenable to MS-based sequence analysis. Here, we showed that the uridine-specific human endoribonuclease hRNase 4 improves mRNA sequence coverage, in comparison with the benchmark enzyme RNase T1, by producing a large population of uniquely mappable cleavage products. Furthermore, we deployed hRNase 4 to characterize mRNAs fully substituted with 1-methylpseudouridine (m1Ψ) or 5-methoxyuridine (mo5U), as well as mRNAs selectively depleted of uridine–two key strategies to reduce synthetic mRNA immunogenicity. Lastly, we demonstrated that hRNase 4 enables direct assessment of the 5′ cap incorporation in in vitro synthesized mRNA. Collectively, this study highlights the power of hRNase 4 to interrogate mRNA sequence, identity, and modifications by LC-MS/MS.

2022-07-26 | PXD033334 | Pride

Genome-wide profiling of DNA methylation in two Arabidopsis ecotypes and their reciprocal hybrids - small RNA-seq

Project description:Heterosis is a fundamental biological phenomenon characterized by the superior performance of a hybrid over its parents in many traits, but the underlying molecular basis remains elusive. To investigate whether DNA methylation plays a role in heterosis, we compared at single base-pair resolution the DNA methylomes of Arabidopsis Ler and C24 parental lines and their reciprocal F1 hybrids that exhibited heterosis for many quantitative traits. Both hybrids displayed increased DNA methylation across their entire genomes, especially in transposable elements. Interestingly, we found that increased methylation of the hybrid genomes predominantly occurred in regions that were differentially methylated in the two parents and covered by small RNAs (sRNAs), implying that the RNA-directed DNA methylation (RdDM) pathway may direct DNA methylation in hybrids. In addition, we found that 77 genes sensitive to remodeling of DNA methylation were transcriptionally repressed in both reciprocal hybrids, including genes involved in flavonoid biosynthesis and two circadian oscillator genes, CIRCADIAN CLOCK ASSOCIATED1 and LATE ELONGATED HYPOCOTYL. Moreover, growth vigor of F1 hybrids was compromised by treatment with an agent that demethylates DNA, and by abolishing production of functional small RNAs due to mutations in Arabidopsis RNA methyltransferase HUA ENHANCER1. Together, our data suggest that genome-wide remodeling of DNA methylation directed by the RdDM pathway may play a role in hybrid vigor. Examination of small RNA sequencing in 2 Arabidopsis ecotypes and their reciprocal hybrids.

2011-12-22 | E-GEOD-34656 | biostudies-arrayexpress

NSun2-mediated cytosine-5 methylation in Vault non-coding RNA determines its processing into small RNAs [iCLIP]

Project description:Autosomal-recessive loss of the NSUN2 gene has been recently identified as a causative link to intellectual disability disorders in humans. NSun2 is an RNA methyltransferase modifying cytosine-5 in transfer RNAs (tRNA). Whether NSun2 methylates additional RNA species is currently debated. Here, we adapted the individual-nucleotide resolution UV cross-linking and immunoprecipitation method (iCLIP) to identify NSun2-mediated methylation in RNA transcriptome. We confirm site-specific methylation in tRNA and identify messenger and non-coding RNAs as potential methylation targets for NSun2. Using RNA bisulfite sequencing we establish Vault non-coding RNAs as novel substrates for NSun2 and identified six cytosine-5 methylated sites. Furthermore, we show that loss of cytosine-5 methylation in Vault RNAs causes aberrant processing into argonaute-associating small RNA fragments (svRNA). Thus, impaired Vault non-coding RNA processing may be an important contributor to the etiology of NSUN2-deficieny human disorders. Identification of Nsun2 targets by miCLIP in Embryonic kidney (HEK293) cells

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The coding and noncoding architecture of the Caulobacter crescentus genome

Project description:Caulobacter crescentus undergoes an asymmetric cell division controlled by a genetic circuit that cycles in space and time. We provide a universal strategy for defining the coding potential of bacterial genomes by applying ribosome profiling, RNA-seq, global 5’-RACE, and liquid chromatography coupled with tandem mass spectrometry (LC-MS) data to the 4-megabase C. crescentus genome. We mapped transcript units at single base-pair resolution using RNA-seq together with global 5’ RACE. Additionally, using ribosome profiling and LC-MS, we mapped translation start sites and coding regions with near complete coverage. We found most start codons lacked corresponding Shine-Dalgarno sites although ribosomes were observed to pause at internal Shine-Dalgarno sites within the ORF. These data suggest a more prevalent use of the Shine-Dalgarno sequence for ribosome pausing rather than translation initiation in C. crescentus. Overall 19% of the transcribed and translated genomic elements were newly identified or significantly improved by this approach providing a valuable genomic resource to elucidate the complete C. crescentus genetic circuitry that controls asymmetric cell division. Ribosome profiling and RNA-seq data were collected in Caulobacter crescentus NA1000 cells grown in M2G and PYE media to map transcript and ORF features in the genome.

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