scholarly journals qDRIP: Quantitative differential RNA:DNA hybrid immunoprecipitation sequencing

2019 ◽  
Author(s):  
Madzia P Crossley ◽  
Michael J Bocek ◽  
Stephan Hamperl ◽  
Tomek Swigut ◽  
Karlene A. Cimprich
Keyword(s):  
Absolute Count ◽  
Internal Standards ◽  
Physiological Processes ◽  
Quantitative Measurements ◽  
Genome Wide ◽  
Accurate Normalization ◽  
Rna:Dna Hybrid ◽  
Rna:Dna Hybrids ◽  
Nucleic Acid Structures ◽  
Generation Sequencing

AbstractR-loops are dynamic, co-transcriptional nucleic acid structures that facilitate physiological processes and cause DNA damage in certain contexts. Perturbations of transcription or R-loop resolution are expected to change their genomic distribution. Next-generation sequencing approaches to map RNA:DNA hybrids, a component of R-loops, have so far not allowed quantitative comparisons between such conditions. Here we describe quantitative differential RNA:DNA immunoprecipitation (qDRIP), a method combining synthetic RNA:DNA-hybrid internal standards with high-resolution, strand-specific sequencing. We show that qDRIP avoids biases inherent to read-count normalization by accurately profiling signal in regions unaffected by transcription inhibition in human cells, and by facilitating accurate differential peak calling between conditions. Finally, we use these quantitative comparisons to make the first estimates of the absolute count of RNA:DNA hybrids per cell and their half-lives genome-wide. Overall, qDRIP allows for accurate normalization in conditions where R-loops are perturbed and for quantitative measurements that provide previously unattainable biological insights.

scholarly journals qDRIP: a method to quantitatively assess RNA–DNA hybrid formation genome-wide

2020 ◽  
Vol 48 (14) ◽  
pp. e84-e84 ◽  
Author(s):  
Magdalena P Crossley ◽  
Michael J Bocek ◽  
Stephan Hamperl ◽  
Tomek Swigut ◽  
Karlene A Cimprich
Keyword(s):  
Rnase H ◽  
Internal Standards ◽  
Physiological Processes ◽  
Quantitative Measurements ◽  
Genome Wide ◽  
Gc Skew ◽  
Accurate Normalization ◽  
Rna Immunoprecipitation ◽  
Nucleic Acid Structures ◽  
Generation Sequencing

Abstract R-loops are dynamic, co-transcriptional nucleic acid structures that facilitate physiological processes but can also cause DNA damage in certain contexts. Perturbations of transcription or R-loop resolution are expected to change their genomic distribution. Next-generation sequencing approaches to map RNA–DNA hybrids, a component of R-loops, have so far not allowed quantitative comparisons between such conditions. Here, we describe quantitative differential DNA–RNA immunoprecipitation (qDRIP), a method combining synthetic RNA–DNA-hybrid internal standards with high-resolution, strand-specific sequencing. We show that qDRIP avoids biases inherent to read-count normalization by accurately profiling signal in regions unaffected by transcription inhibition in human cells, and by facilitating accurate differential peak calling between conditions. We also use these quantitative comparisons to make the first estimates of the absolute count of RNA–DNA hybrids per cell and their half-lives genome-wide. Finally, we identify a subset of RNA–DNA hybrids with high GC skew which are partially resistant to RNase H. Overall, qDRIP allows for accurate normalization in conditions where R-loops are perturbed and for quantitative measurements that provide previously unattainable biological insights.

scholarly journals Pathogenic mutations reveal a role of RECQ4 in mitochondrial RNA:DNA hybrid formation and resolution

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Chou-Wei Chang ◽  
Xiaohua Xu ◽  
Min Li ◽  
Di Xin ◽  
Lin Ding ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
Biochemical Analysis ◽  
Mitochondrial Localization ◽  
Protein Protein Interaction ◽  
Complex Process ◽  
Rna:Dna Hybrid ◽  
Rna:Dna Hybrids ◽  
Nucleic Acid Structures ◽  
Mitochondrial Membrane Protein

Abstract The synthesis of mitochondrial DNA (mtDNA) is a complex process that involves the formation and resolution of unusual nucleic acid structures, such as RNA:DNA hybrids. However, little is known about the enzymes that regulate these processes. RECQ4 is a DNA replication factor important for mtDNA maintenance, and here, we unveil a role of human RECQ4 in regulating the formation and resolution of mitochondrial RNA:DNA hybrids. Mitochondrial membrane protein p32 can block mtDNA synthesis by restricting RECQ4 mitochondrial localization via protein–protein interaction. We found that the interaction with p32 was disrupted not only by the previously reported cancer-associated RECQ4 mutation, del(A420-A463), but also by a clinical mutation of the adjacent residue, P466L. Surprisingly, although P466L mutant was present in the mitochondria at greater levels, unlike del(A420-A463) mutant, it failed to enhance mtDNA synthesis due to the accumulation of RNA:DNA hybrids throughout the mtDNA. Biochemical analysis revealed that P466L mutation enhanced RECQ4 annealing activity to generate RNA:DNA hybrids at the same time reduced its unwinding activity to resolve this structure. Hence, P466L mutation led to a reduced efficiency in completing mtDNA synthesis due to unresolved RNA:DNA hybrids across mtDNA.

scholarly journals Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

2021 ◽  
Vol 220 (6) ◽  
Author(s):  
John A. Smolka ◽  
Lionel A. Sanz ◽  
Stella R. Hartono ◽  
Frédéric Chédin
Keyword(s):  
Ribosomal Rna ◽  
Rna Binding ◽  
Binding Activity ◽  
Genome Wide ◽  
Additional Imaging ◽  
Rna:Dna Hybrid ◽  
Rna:Dna Hybrids ◽  
The Impact ◽  
Target Rna

The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid–specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.

scholarly journals Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi–Goutières syndrome

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Yoong Wearn Lim ◽  
Lionel A Sanz ◽  
Xiaoqin Xu ◽  
Stella R Hartono ◽  
Frédéric Chédin
Keyword(s):  
Nucleic Acid ◽  
Lupus Erythematosus ◽  
Inflammatory Disorder ◽  
Dna Hypomethylation ◽  
Systemic Lupus ◽  
Genome Wide ◽  
Genetic Overlap ◽  
Rna:Dna Hybrid ◽  
Rna:Dna Hybrids ◽  
Rnase H2

Aicardi–Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE). AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4), and SAMHD1 (AGS5). However, the identity and source of such immunogenic nucleic acid species remain undefined. Using genome-wide approaches, we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids. Using MethylC-seq, we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions. Altogether, our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS, furthering the links between AGS and SLE.

scholarly journals Large-scale comparative epigenomics reveals hierarchical regulation of non-CG methylation in Arabidopsis

2018 ◽  
Vol 115 (5) ◽  
pp. E1069-E1074 ◽  
Author(s):  
Yu Zhang ◽  
C. Jake Harris ◽  
Qikun Liu ◽  
Wanlu Liu ◽  
Israel Ausin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Large Scale ◽  
Bisulfite Conversion ◽  
Quantitative Measurements ◽  
Comparative Epigenomics ◽  
Genome Wide ◽  
Genome Bisulfite Sequencing ◽  
Tremendous Amount ◽  
Dna Methylation Analysis ◽  
Generation Sequencing

Genome-wide characterization by next-generation sequencing has greatly improved our understanding of the landscape of epigenetic modifications. Since 2008, whole-genome bisulfite sequencing (WGBS) has become the gold standard for DNA methylation analysis, and a tremendous amount of WGBS data has been generated by the research community. However, the systematic comparison of DNA methylation profiles to identify regulatory mechanisms has yet to be fully explored. Here we reprocessed the raw data of over 500 publicly available Arabidopsis WGBS libraries from various mutant backgrounds, tissue types, and stress treatments and also filtered them based on sequencing depth and efficiency of bisulfite conversion. This enabled us to identify high-confidence differentially methylated regions (hcDMRs) by comparing each test library to over 50 high-quality wild-type controls. We developed statistical and quantitative measurements to analyze the overlapping of DMRs and to cluster libraries based on their effect on DNA methylation. In addition to confirming existing relationships, we revealed unanticipated connections between well-known genes. For instance, MET1 and CMT3 were found to be required for the maintenance of asymmetric CHH methylation at nonoverlapping regions of CMT2 targeted heterochromatin. Our comparative methylome approach has established a framework for extracting biological insights via large-scale comparison of methylomes and can also be adopted for other genomics datasets.

scholarly journals Genome-wide discovery of G-quadruplexes in barley

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
H. Busra Cagirici ◽  
Hikmet Budak ◽  
Taner Z. Sen
Keyword(s):  
Transcription Initiation ◽  
Large Window ◽  
First Intron ◽  
Domain Sequence ◽  
Genome Wide ◽  
Square Planar ◽  
Peak Points ◽  
Molecular Functions ◽  
Nucleic Acid Structures ◽  
Provided Examples

AbstractG-quadruplexes (G4s) are four-stranded nucleic acid structures with closely spaced guanine bases forming square planar G-quartets. Aberrant formation of G4 structures has been associated with genomic instability. However, most plant species are lacking comprehensive studies of G4 motifs. In this study, genome-wide identification of G4 motifs in barley was performed, followed by a comparison of genomic distribution and molecular functions to other monocot species, such as wheat, maize, and rice. Similar to the reports on human and some plants like wheat, G4 motifs peaked around the 5′ untranslated region (5′ UTR), the first coding domain sequence, and the first intron start sites on antisense strands. Our comparative analyses in human, Arabidopsis, maize, rice, and sorghum demonstrated that the peak points could be erroneously merged into a single peak when large window sizes are used. We also showed that the G4 distributions around genic regions are relatively similar in the species studied, except in the case of Arabidopsis. G4 containing genes in monocots showed conserved molecular functions for transcription initiation and hydrolase activity. Additionally, we provided examples of imperfect G4 motifs.

scholarly journals Identification, Expression and Evolution of Short-Chain Dehydrogenases/Reductases in Nile Tilapia (Oreochromis niloticus)

2021 ◽  
Vol 22 (8) ◽  
pp. 4201
Author(s):  
Shuai Zhang ◽  
Lang Xie ◽  
Shuqing Zheng ◽  
Baoyue Lu ◽  
Wenjing Tao ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Tandem Duplication ◽  
Developmental Stages ◽  
Short Chain ◽  
Sexually Dimorphic ◽  
Physiological Processes ◽  
Genome Wide ◽  
Nile Tilapia Oreochromis Niloticus ◽  
Tandem Duplications

The short-chain dehydrogenases/reductases (SDR) superfamily is involved in multiple physiological processes. In this study, genome-wide identification and comprehensive analysis of SDR superfamily were carried out in 29 animal species based on the latest genome databases. Overall, the number of SDR genes in animals increased with whole genome duplication (WGD), suggesting the expansion of SDRs during evolution, especially in 3R-WGD and polyploidization of teleosts. Phylogenetic analysis indicated that vertebrates SDRs were clustered into five categories: classical, extended, undefined, atypical, and complex. Moreover, tandem duplication of hpgd-a, rdh8b and dhrs13 was observed in teleosts analyzed. Additionally, tandem duplications of dhrs11-a, dhrs7a, hsd11b1b, and cbr1-a were observed in all cichlids analyzed, and tandem duplication of rdh10-b was observed in tilapiines. Transcriptome analysis of adult fish revealed that 93 SDRs were expressed in more than one tissue and 5 in one tissue only. Transcriptome analysis of gonads from different developmental stages showed that expression of 17 SDRs were sexually dimorphic with 11 higher in ovary and 6 higher in testis. The sexually dimorphic expressions of these SDRs were confirmed by in situ hybridization (ISH) and qPCR, indicating their possible roles in steroidogenesis and gonadal differentiation. Taken together, the identification and the expression data obtained in this study contribute to a better understanding of SDR superfamily evolution and functions in teleosts.

scholarly journals Genome-wide identification and analysis of class III peroxidases in Betula pendula

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Kewei Cai ◽  
Huixin Liu ◽  
Song Chen ◽  
Yi Liu ◽  
Xiyang Zhao ◽  
...  
Keyword(s):  
Stress Responses ◽  
Betula Pendula ◽  
Expression Patterns ◽  
Class Iii ◽  
Physiological Processes ◽  
Plant Life ◽  
Genome Wide ◽  
Plant Life Cycle ◽  
Class Iii Peroxidases ◽  
And Function

Abstract Background Class III peroxidases (POD) proteins are widely present in the plant kingdom that are involved in a broad range of physiological processes including stress responses and lignin polymerization throughout the plant life cycle. At present, POD genes have been studied in Arabidopsis, rice, poplar, maize and Chinese pear, but there are no reports on the identification and function of POD gene family in Betula pendula. Results We identified 90 nonredundant POD genes in Betula pendula. (designated BpPODs). According to phylogenetic relationships, these POD genes were classified into 12 groups. The BpPODs are distributed in different numbers on the 14 chromosomes, and some BpPODs were located sequentially in tandem on chromosomes. In addition, we analyzed the conserved domains of BpPOD proteins and found that they contain highly conserved motifs. We also investigated their expression patterns in different tissues, the results showed that some BpPODs might play an important role in xylem, leaf, root and flower. Furthermore, under low temperature conditions, some BpPODs showed different expression patterns at different times. Conclusions The research on the structure and function of the POD genes in Betula pendula plays a very important role in understanding the growth and development process and the molecular mechanism of stress resistance. These results lay the theoretical foundation for the genetic improvement of Betula pendula.

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