Genome-wide profiling of in vivo RNA structure at single-nucleotide resolution using structure-seq

2015 ◽  
Vol 10 (7) ◽  
pp. 1050-1066 ◽  
Author(s):  
Yiliang Ding ◽  
Chun Kit Kwok ◽  
Yin Tang ◽  
Philip C Bevilacqua ◽  
Sarah M Assmann
Keyword(s):  
Rna Structure ◽  
Single Nucleotide ◽  
Genome Wide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

scholarly journals FICC-Seq: a method for enzyme-specified profiling of methyl-5-uridine in cellular RNA

2019 ◽  
Vol 47 (19) ◽  
pp. e113-e113 ◽  
Author(s):  
Jean-Michel Carter ◽  
Warren Emmett ◽  
Igor Rdl Mozos ◽  
Annika Kotter ◽  
Mark Helm ◽  
...  
Keyword(s):  
Mapping Method ◽  
Single Nucleotide ◽  
Genome Wide ◽  
A Genome ◽  
Target Sites ◽  
Reliable Detection ◽  
Resolution Mapping ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

Abstract Methyl-5-uridine (m5U) is one the most abundant non-canonical bases present in cellular RNA, and in yeast is found at position U54 of tRNAs where modification is catalysed by the methyltransferase Trm2. Although the mammalian enzymes that catalyse m5U formation are yet to be identified via experimental evidence, based on sequence homology to Trm2, two candidates currently exist, TRMT2A and TRMT2B. Here we developed a genome-wide single-nucleotide resolution mapping method, Fluorouracil-Induced-Catalytic-Crosslinking-Sequencing (FICC-Seq), in order to identify the relevant enzymatic targets. We demonstrate that TRMT2A is responsible for the majority of m5U present in human RNA, and that it commonly targets U54 of cytosolic tRNAs. By comparison to current methods, we show that FICC-Seq is a particularly robust method for accurate and reliable detection of relevant enzymatic target sites. Our associated finding of extensive irreversible TRMT2A-tRNA crosslinking in vivo following 5-Fluorouracil exposure is also intriguing, as it suggests a tangible mechanism for a previously suspected RNA-dependent route of Fluorouracil-mediated cytotoxicity.

scholarly journals Mapping ribonucleotides embedded in genomic DNA to single-nucleotide resolution using Ribose-Map

2020 ◽  
Author(s):  
Alli L. Gombolay ◽  
Francesca Storici
Keyword(s):  
Computing Time ◽  
Wide Distribution ◽  
Sequencing Analysis ◽  
Sequencing Data ◽  
Single Nucleotide ◽  
Genome Wide ◽  
Hands On ◽  
Nucleotide Resolution ◽  
User Friendly ◽  
Single Nucleotide Resolution

ABSTRACTRibose-Map is a user-friendly, standardized bioinformatics toolkit for the comprehensive analysis of ribonucleotide sequencing experiments. It allows researchers to map the locations of ribonucleotides in DNA to single-nucleotide resolution and identify biological signatures of ribonucleotide incorporation. In addition, it can be applied to data generated using any currently available high-throughput ribonucleotide sequencing technique, thus standardizing the analysis of ribonucleotide sequencing experiments and allowing direct comparisons of results. This protocol describes in detail how to use Ribose-Map to analyze raw ribonucleotide sequencing data, including preparing the reads for analysis, locating the genomic coordinates of ribonucleotides, exploring the genome-wide distribution of ribonucleotides, determining the nucleotide sequence context of ribonucleotides, and identifying hotspots of ribonucleotide incorporation. Ribose-Map does not require background knowledge of ribonucleotide sequencing analysis and assumes only basic command-line skills. The protocol requires less than 3 hr of computing time for most datasets and about 30 min of hands-on time.

scholarly journals Single-nucleotide resolution dynamic repair maps of UV damage in Saccharomyces cerevisiae genome

2018 ◽  
Vol 115 (15) ◽  
pp. E3408-E3415 ◽  
Author(s):  
Wentao Li ◽  
Ogun Adebali ◽  
Yanyan Yang ◽  
Christopher P. Selby ◽  
Aziz Sancar
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Excision Repair ◽  
Model Organism ◽  
Early Time ◽  
Uv Damage ◽  
Single Nucleotide ◽  
Pyrimidine Dimers ◽  
Genome Wide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

We have adapted the eXcision Repair-sequencing (XR-seq) method to generate single-nucleotide resolution dynamic repair maps of UV-induced cyclobutane pyrimidine dimers and (6-4) pyrimidine–pyrimidone photoproducts in the Saccharomyces cerevisiae genome. We find that these photoproducts are removed from the genome primarily by incisions 13–18 nucleotides 5′ and 6–7 nucleotides 3′ to the UV damage that generate 21- to 27-nt-long excision products. Analyses of the excision repair kinetics both in single genes and at the genome-wide level reveal strong transcription-coupled repair of the transcribed strand at early time points followed by predominantly nontranscribed strand repair at later stages. We have also characterized the excision repair level as a function of the transcription level. The availability of high-resolution and dynamic repair maps should aid in future repair and mutagenesis studies in this model organism.

scholarly journals CNCC: an analysis tool to determine genome-wide DNA break end structure at single-nucleotide resolution

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Karol Szlachta ◽  
Heather M. Raimer ◽  
Laurey D. Comeau ◽  
Yuh-Hwa Wang
Keyword(s):  
Mapping Technique ◽  
Nucleotide Position ◽  
Analysis Tool ◽  
Single Nucleotide ◽  
Genome Wide ◽  
A Genome ◽  
Cross Correlation Analysis ◽  
Dna Break ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

Abstract Background DNA double-stranded breaks (DSBs) are potentially deleterious events in a cell. The end structures (blunt, 3′- and 5′-overhangs) at DSB sites contribute to the fate of their repair and provide critical information concerning the consequences of the damage. Therefore, there has been a recent eruption of DNA break mapping and sequencing methods that aim to map at single-nucleotide resolution where breaks are generated genome-wide. These methods provide high resolution data for the location of DSBs, which can encode the type of end-structure present at these breaks. However, genome-wide analysis of the resulting end structures has not been investigated following these sequencing methods. Results To address this analysis gap, we develop the use of a coverage-normalized cross correlation analysis (CNCC) to process the high-precision genome-wide break mapping data, and determine genome-wide break end structure distributions at single-nucleotide resolution. We take advantage of the single-nucleotide position and the knowledge of strandness from every mapped break to analyze the relative shifts between positive and negative strand encoded break nucleotides. By applying CNCC we can identify the most abundant end structures captured by a break mapping technique, and further can make comparisons between different samples and treatments. We validate our analysis with restriction enzyme digestions of genomic DNA and establish the sensitivity of the analysis using end structures that only exist as a minor fraction of total breaks. Finally, we demonstrate the versatility of our analysis by applying CNCC to the breaks resulting after treatment with etoposide and study the variety of resulting end structures. Conclusion For the first time, on a genome-wide scale, our analysis revealed the increase in the 5′ to 3′ end resection following etoposide treatment, and the global progression of the resection. Furthermore, our method distinguished the change in the pattern of DSB end structure with increasing doses of the drug. The ability of this method to determine DNA break end structures without a priori knowledge of break sequences or genomic position should have broad applications in understanding genome instability.

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