Detection of RNA Editing Events in Human Cells Using High-Throughput Sequencing

Adenosine-to-inosine RNA editing may be implicated in human pathogenesis

Bulletin of Russian State Medical University ◽

10.24075/brsmu.2019.028 ◽

2019 ◽

pp. 22-25

Author(s):

AA Kliuchnikova ◽

SA Moshkovskii

Keyword(s):

Immune Responses ◽

Rna Editing ◽

High Throughput ◽

High Throughput Sequencing ◽

Common Mechanism ◽

Adenosine Deaminases ◽

Human Transcriptome ◽

Sequencing Technologies

Adenosine-to-inosine (A-to-I) RNA editing is a common mechanism of post-transcriptional modification in many metazoans including vertebrates; the process is catalyzed by adenosine deaminases acting on RNA (ADARs). Using high-throughput sequencing technologies resulted in finding thousands of RNA editing sites throughout the human transcriptome however, their functions are still poorly understood. The aim of this brief review is to draw attention of clinicians and biomedical researchers to ADAR-mediated RNA editing phenomenon and its possible implication in development of neuropathologies, antiviral immune responses and cancer.

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Intrinsic and regulated properties of minimally edited trypanosome mRNAs

Nucleic Acids Research ◽

10.1093/nar/gkz012 ◽

2019 ◽

Vol 47 (7) ◽

pp. 3640-3657 ◽

Cited By ~ 7

Author(s):

Brianna L Tylec ◽

Rachel M Simpson ◽

Laura E Kirby ◽

Runpu Chen ◽

Yijun Sun ◽

...

Keyword(s):

Rna Editing ◽

High Throughput ◽

High Throughput Sequencing ◽

Substrate Binding ◽

Open Reading Frames ◽

Mrna Editing ◽

Accessory Factors ◽

Reading Frames ◽

Complex Components

Abstract Most mitochondrial mRNAs in kinetoplastids require extensive uridine insertion/deletion editing to generate translatable open reading frames. Editing is specified by trans-acting gRNAs and involves a complex machinery including basal and accessory factors. Here, we utilize high-throughput sequencing to analyze editing progression in two minimally edited mRNAs that provide a simplified system due their requiring only two gRNAs each for complete editing. We show that CYb and MURF2 mRNAs exhibit barriers to editing progression that differ from those previously identified for pan-edited mRNAs, primarily at initial gRNA usage and gRNA exchange. We demonstrate that mis-edited junctions arise through multiple pathways including mis-alignment of cognate gRNA, incorrect and sometimes promiscuous gRNA utilization and inefficient gRNA anchoring. We then examined the roles of accessory factors RBP16 and MRP1/2 in maintaining edited CYb and MURF2 populations. RBP16 is essential for initiation of CYb and MURF2 editing, as well as MURF2 editing progression. In contrast, MRP1/2 stabilizes both edited mRNA populations, while further promoting progression of MURF2 mRNA editing. We also analyzed the effects of RNA Editing Substrate Binding Complex components, TbRGG2 and GAP1, and show that both proteins modestly impact progression of editing on minimally edited mRNAs, suggesting a novel function for GAP1.

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Abstract 59: Identification of RNA editing events in cancer using high-throughput sequencing data

10.1158/1538-7445.am2011-59 ◽

2011 ◽

Author(s):

Amie Radenbaugh ◽

John Z. Sanborn ◽

Daniel Zerbino ◽

Chris Wilks ◽

Joshua M. Stuart ◽

...

Keyword(s):

Rna Editing ◽

High Throughput ◽

High Throughput Sequencing ◽

Sequencing Data ◽

High Throughput Sequencing Data

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Rapid high-resolution measurement of DNA replication timing by droplet digital PCR

10.1101/208546 ◽

2017 ◽

Author(s):

Dzmitry G. Batrakou ◽

Emma D. Heron ◽

Conrad A. Nieduszynski

Keyword(s):

Dna Replication ◽

High Throughput ◽

High Throughput Sequencing ◽

Replication Timing ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Human Cells ◽

Genome Replication ◽

Multiple Loci ◽

Dna Replication Timing

ABSTRACTGenomes are replicated in a reproducible temporal pattern. Current methods for assaying allele replication timing are time consuming and/or expensive. These include high-throughput sequencing which can be used to measure DNA copy number as a proxy for allele replication timing. Here, we use droplet digital PCR to study DNA replication timing at multiple loci in budding yeast and human cells. We establish that the method has temporal and spatial resolutions comparable to the high-throughput sequencing approaches, while being faster than alternative locus-specific methods. Furthermore, the approach is capable of allele discrimination. We apply this method to determine relative replication timing across timing transition zones in cultured human cells. Finally, multiple samples can be analysed in parallel, allowing us to rapidly screen kinetochore mutants for perturbation to centromere replication timing. Therefore, this approach is well suited to the study of locus-specific replication and the screening of cis- and trans-acting mutants to identify mechanisms that regulate local genome replication timing.

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Genome-wide identification of RNA editing in seven porcine tissues by matched DNA and RNA high-throughput sequencing

Journal of Animal Science and Biotechnology ◽

10.1186/s40104-019-0326-9 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 5

Author(s):

Yuebo Zhang ◽

Longchao Zhang ◽

Jingwei Yue ◽

Xia Wei ◽

Ligang Wang ◽

...

Keyword(s):

Rna Editing ◽

High Throughput ◽

High Throughput Sequencing ◽

Dna And Rna ◽

Genome Wide ◽

Porcine Tissues

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Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1517883113 ◽

2016 ◽

Vol 113 (9) ◽

pp. 2544-2549 ◽

Cited By ~ 122

Author(s):

Alan S. L. Wong ◽

Gigi C. G. Choi ◽

Cheryl H. Cui ◽

Gabriela Pregernig ◽

Pamela Milani ◽

...

Keyword(s):

Ovarian Cancer ◽

High Throughput ◽

High Throughput Sequencing ◽

Drug Combinations ◽

Human Cells ◽

Ovarian Cancer Cells ◽

Specific Gene ◽

Cancer Cell Growth ◽

Guide Rna ◽

Systematic Identification

The orchestrated action of genes controls complex biological phenotypes, yet the systematic discovery of gene and drug combinations that modulate these phenotypes in human cells is labor intensive and challenging to scale. Here, we created a platform for the massively parallel screening of barcoded combinatorial gene perturbations in human cells and translated these hits into effective drug combinations. This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (CombiGEM) to rapidly assemble barcoded combinatorial genetic libraries that can be tracked with high-throughput sequencing. We applied CombiGEM-CRISPR to create a library of 23,409 barcoded dual guide-RNA (gRNA) combinations and then perform a high-throughput pooled screen to identify gene pairs that inhibited ovarian cancer cell growth when they were targeted. We validated the growth-inhibiting effects of specific gene sets, including epigenetic regulators KDM4C/BRD4 and KDM6B/BRD4, via individual assays with CRISPR-Cas–based knockouts and RNA-interference–based knockdowns. We also tested small-molecule drug pairs directed against our pairwise hits and showed that they exerted synergistic antiproliferative effects against ovarian cancer cells. We envision that the CombiGEM-CRISPR platform will be applicable to a broad range of biological settings and will accelerate the systematic identification of genetic combinations and their translation into novel drug combinations that modulate complex human disease phenotypes.

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