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 An RNA-binding switch drives ribosome biogenesis and tumorigenesis downstream of RAS oncogene

2021 ◽  
Author(s):  
Muhammad S Azman ◽  
Martin Dodel ◽  
Federica Capraro ◽  
Rupert Faraway ◽  
Maria Dermit ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ribosomal Rna ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Binding Activity ◽  
Ras Signaling ◽  
Cancer Cell Proliferation ◽  
Inhibit Tumor Growth ◽  
Oncogenic Ras

Oncogenic RAS signaling reprograms gene expression through both transcriptional and post-transcriptional mechanisms. While transcriptional regulation downstream of RAS is relatively well-characterized, how RAS post-transcriptionally modulates gene expression to promote malignancy is unclear. Using quantitative RNA Interactome Capture analysis, we reveal that oncogenic RAS signaling reshapes the RNA-bound proteomic landscape of cancer cells, with a network of nuclear proteins centered around Nucleolin displaying enhanced RNA-binding activity. We show that Nucleolin is phosphorylated downstream of RAS, which increases its binding to pre-ribosomal-RNA (rRNA), boosts rRNA production, and promotes ribosome biogenesis. This Nucleolin-dependent enhancement of ribosome biogenesis is crucial for RAS-induced cancer cell proliferation, and can be targeted therapeutically to inhibit tumor growth. Our results reveal that oncogenic RAS signaling drives ribosome biogenesis by regulating the RNA-binding activity of Nucleolin, and highlights the crucial role of this process in RAS-mediated tumorigenesis.

­Dysregulation of the sensory and regulatory pathways controlling cellular iron metabolism in unilateral obstructive nephropathy

2021 ◽  
Author(s):  
James A Votava ◽  
Shannon Reese ◽  
Kathryn M Deck ◽  
Christopher P Nizzi ◽  
Sheila Anderson ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Kidney Failure ◽  
Rna Binding ◽  
Binding Activity ◽  
Iron Storage ◽  
Iron Regulatory Proteins ◽  
Regulatory Pathways ◽  
Genetic Ablation ◽  
Cellular Iron ◽  
The Impact

Chronic kidney disease (CKD) involves disturbances in iron metabolism including anemia caused by insufficient erythropoietin (EPO) production. However, underlying mechanisms responsible for the dysregulation of cellular iron metabolism are incompletely defined. Using the unilateral ureteral obstruction (UUO) model in Irp1+/+ and Irp1-/- mice we asked if iron regulatory proteins (IRP), the central regulators of cellular iron metabolism and also suppressors of EPO production, contribute to the etiology of anemia in kidney failure. We identified a significant reduction in IRP protein level and RNA binding activity that associated with a loss of the iron uptake protein transferrin receptor 1, increased expression of the iron storage protein subunits H- and L-ferritin, and a low but overall variable level of stainable iron in the obstructed kidney. This reduction in IRP RNA binding activity and ferritin RNA levels suggests the concomitant rise in ferritin expression and iron content in kidney failure is IRP-dependent. In contrast, the reduction in Epo mRNA level in the obstructed kidney was not rescued by genetic ablation of IRP1 suggesting disruption of normal HIF-2a regulation. Furthermore, reduced expression of some HIFa target genes in UUO occurred in the face of increased expression of HIFa proteins and the prolyl hydroxylases (PHD) 2 and PHD1, the latter of which is not known to be HIFa mediated. Our results suggest that the IRP system drives changes in cellular iron metabolism that are associated with kidney failure in UUO but that the impact of IRP on EPO production is overridden by disrupted hypoxia signaling.

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 DDX17 is required for efficient DSB repair at DNA:RNA hybrid deficient loci

2021 ◽  
Author(s):  
Aldo S Bader ◽  
Janna Luessing ◽  
Ben R Hawley ◽  
George L Skalka ◽  
Wei-Ting Lu ◽  
...  
Keyword(s):  
Dna Repair ◽  
Rna Binding ◽  
Meta Analysis ◽  
Binding Activity ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks ◽  
Proteomic Data ◽  
Genome Wide ◽  
Repair Factor

Proteins with RNA-binding activity are increasingly being implicated in DNA damage responses (DDR). Additionally, DNA:RNA-hybrids are rapidly generated around DNA double-strand breaks (DSBs), and are essential for effective repair. Here, using a meta-analysis of proteomic data, we identify novel DNA repair proteins and characterise a novel role for DDX17 in DNA repair. We found DDX17 to be required for both cell survival and DNA repair in response to numerous agents that induce DSBs. Analysis of DSB repair factor recruitment to damage sites suggested a role for DDX17 early in the DSB ubiquitin cascade. Genome-wide mapping of R-loops revealed that while DDX17 promotes the formation of DNA:RNA-hybrids around DSB sites, this role is specific to loci that are naturally deficient for DNA:RNA-hybrids. We propose that DDX17 facilitates DSB repair at loci that are inefficient at forming DNA:RNA-hybrids by catalysing the formation of DSB-induced hybrids, thereby allowing propagation of the damage response.

High-fidelity base editor with no detectable genome-wide off-target effects

2020 ◽  
Author(s):  
Erwei Zuo ◽  
Yidi Sun ◽  
Tanglong Yuan ◽  
Bingbing He ◽  
Changyang Zhou ◽  
...  
Keyword(s):  
Rna Binding ◽  
High Fidelity ◽  
Sequencing Analysis ◽  
Single Nucleotide ◽  
Dna And Rna ◽  
Genome Wide ◽  
Cell Embryo ◽  
Single Nucleotide Variations ◽  
Target Rna ◽  
Target Effects

Base editors hold promise for correcting pathogenic mutations, while substantial single nucleotide variations (SNVs) on both DNA and RNA were generated by cytosine base editors (CBEs). Here we examined possibilities to reduce off-target effects by engineering cytosine deaminases. By screening 24 CBEs harboring various rAPOBEC1 (BE3) or human APOBEC3A (BE3-hA3A) mutations on the ssDNA or RNA binding domain, we found 8 CBE variations could maintain high on-target editing efficiency. Using Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI) method and RNA sequencing analysis, we found DNA off-target SNVs induced by BE3 could be completely eliminated in BE3R126E but the off-target RNA SNVs was only slightly reduced. By contrast, BE3-hA3AY130F abolished the RNA off-target effects while could not reduce the DNA off-target effects. Notably, BE3R132E, BE3W90Y+R126E and BE3W90F+R126E achieved the elimination of off-target SNVs on both DNA and RNA, suggesting the feasibility of engineering base editors for high fidelity deaminases.

scholarly journals RNA-Guided Genomic Localization of H2A.L.2 Histone Variant

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 474 ◽  
Author(s):  
Naghmeh Hoghoughi ◽  
Sophie Barral ◽  
Sandrine Curtet ◽  
Florent Chuffart ◽  
Guillaume Charbonnier ◽  
...  
Keyword(s):  
Rna Binding ◽  
Histone Variants ◽  
Binding Activity ◽  
Pericentric Heterochromatin ◽  
Binding Motif ◽  
Genome Wide ◽  
Specific Accumulation ◽  
Open Question ◽  
Genome Scale ◽  
Mature Spermatozoa

The molecular basis of residual histone retention after the nearly genome-wide histone-to-protamine replacement during late spermatogenesis is a critical and open question. Our previous investigations showed that in postmeiotic male germ cells, the genome-scale incorporation of histone variants TH2B-H2A.L.2 allows a controlled replacement of histones by protamines to occur. Here, we highlight the intrinsic ability of H2A.L.2 to specifically target the pericentric regions of the genome and discuss why pericentric heterochromatin is a privileged site of histone retention in mature spermatozoa. We observed that the intranuclear localization of H2A.L.2 is controlled by its ability to bind RNA, as well as by an interplay between its RNA-binding activity and its tropism for pericentric heterochromatin. We identify the H2A.L.2 RNA-binding domain and demonstrate that in somatic cells, the replacement of H2A.L.2 RNA-binding motif enhances and stabilizes its pericentric localization, while the forced expression of RNA increases its homogenous nuclear distribution. Based on these data, we propose that the specific accumulation of RNA on pericentric regions combined with H2A.L.2 tropism for these regions are responsible for stabilizing H2A.L.2 on these regions in mature spermatozoa. This situation would favor histone retention on pericentric heterochromatin.

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 Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

2020 ◽  
Author(s):  
John A. Smolka ◽  
Lionel A. Sanz ◽  
Stella R. Hartono ◽  
Frédéric Chédin
Keyword(s):  
Immunofluorescence Microscopy ◽  
Imaging Data ◽  
Rnase Iii ◽  
Double Stranded Rna ◽  
Hybrid Substrates ◽  
Rna:Dna Hybrid ◽  
Rna:Dna Hybrids ◽  
Pre Treatment

ABSTRACTThe contribution of RNA:DNA hybrid metabolism to cellular processes and disease states has become a prominent topic of study. The S9.6 antibody recognizes RNA:DNA hybrids with a subnanomolar affinity, making it a broadly used tool to detect and study RNA:DNA hybrids. However, S9.6 also binds double-stranded RNA in vitro with significant affinity. Though frequently used in immunofluorescence microscopy, the possible reactivity of S9.6 with non-RNA:DNA hybrid substrates in situ, particularly RNA, has not been comprehensively addressed. Furthermore, S9.6 immunofluorescence microscopy has been methodologically variable and generated discordant imaging datasets. In this study, we find that the majority of the S9.6 immunofluorescence signal observed in fixed human cells arises from RNA, not RNA:DNA hybrids. S9.6 staining was quantitatively unchanged by pre-treatment with the human RNA:DNA hybrid-specific nuclease, RNase H1, despite experimental verification in situ that S9.6 could recognize RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to pre-treatments with RNase T1, and in some cases RNase III, two ribonucleases that specifically degrade single-stranded and double-stranded RNA, respectively. In contrast, genome-wide maps obtained by high-throughput DNA sequencing after S9.6-mediated DNA:RNA Immunoprecipitation (DRIP) are RNase H1-sensitive and RNase T1- and RNase III-insensitive. Altogether, these data demonstrate that the S9.6 antibody, though capable of recognizing RNA:DNA hybrids in situ and in vitro, suffers from a lack of specificity that precludes reliable imaging of RNA:DNA hybrids and renders associated imaging data inconclusive in the absence of controls for its promiscuous recognition of cellular RNAs.

In situ Quantification of the Impact of Episodic Enhanced Turbulent Events in Large Phytoplankton

2011 ◽  
Author(s):  
Percy L. Donaghay ◽  
Jan Rines ◽  
James Sullivan
Keyword(s):  
The Impact
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