scholarly journals Mouse papillomavirus type 1 (MmuPV1) DNA is frequently integrated in benign tumors by microhomology-mediated end-joining

2021 ◽  
Vol 17 (8) ◽  
pp. e1009812
Author(s):  
Lulu Yu ◽  
Vladimir Majerciak ◽  
Xiang-Yang Xue ◽  
Aayushi Uberoi ◽  
Alexei Lobanov ◽  
...  
Keyword(s):  
Single Molecule ◽  
Benign Tumors ◽  
End Joining ◽  
Mitotic Kinases ◽  
Normal Tissues ◽  
E4 Region ◽  
Chimeric Rnas ◽  
Integration Sites ◽  
Intergenic Regions ◽  
Rapid Amplification

MmuPV1 is a useful model for studying papillomavirus-induced tumorigenesis. We used RNA-seq to look for chimeric RNAs that map to both MmuPV1 and host genomes. In tumor tissues, a higher proportion of total viral reads were virus-host chimeric junction reads (CJRs) (1.9‰ - 7‰) than in tumor-free tissues (0.6‰- 1.3‰): most CJRs mapped to the viral E2/E4 region. Although most of the MmuPV1 integration sites were mapped to intergenic regions and introns throughout the mouse genome, integrations were seen more than once in several genes: Malat1, Krt1, Krt10, Fabp5, Pard3, and Grip; these data were confirmed by rapid amplification of cDNA ends (RACE)-Single Molecule Real-Time (SMRT)-seq or targeted DNA-seq. Microhomology sequences were frequently seen at host-virus DNA junctions. MmuPV1 infection and integration affected the expression of host genes. We found that factors for DNA double-stranded break repair and microhomology-mediated end-joining (MMEJ), such as H2ax, Fen1, DNA polymerase Polθ, Cdk1, and Plk1, exhibited a step-wise increase and Mdc1 a decrease in expression in MmuPV1-infected tissues and MmuPV1 tumors relative to normal tissues. Increased expression of mitotic kinases CDK1 and PLK1 appears to be correlated with CtIP phosphorylation in MmuPV1 tumors, suggesting a role for MMEJ-mediated DNA joining in the MmuPV1 integration events that are associated with MmuPV1-induced progression of tumors.

scholarly journals Identification of the cross-strand chimeric RNAs generated by fusions of bi-directional transcripts

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yuting Wang ◽  
Qin Zou ◽  
Fajin Li ◽  
Wenwei Zhao ◽  
Hui Xu ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Sequencing Data ◽  
Normal Tissues ◽  
Rna Transcripts ◽  
Chimeric Rnas ◽  
Dna Strands ◽  
Comprehensive Survey ◽  
The Cross ◽  
Chimeric Rna ◽  
Cancerous Cells

AbstractA major part of the transcriptome complexity is attributed to multiple types of DNA or RNA fusion events, which take place within a gene such as alternative splicing or between different genes such as DNA rearrangement and trans-splicing. In the present study, using the RNA deep sequencing data, we systematically survey a type of non-canonical fusions between the RNA transcripts from the two opposite DNA strands. We name the products of such fusion events cross-strand chimeric RNA (cscRNA). Hundreds to thousands of cscRNAs can be found in human normal tissues, primary cells, and cancerous cells, and in other species as well. Although cscRNAs exhibit strong tissue-specificity, our analysis identifies thousands of recurrent cscRNAs found in multiple different samples. cscRNAs are mostly originated from convergent transcriptions of the annotated genes and their anti-sense DNA. The machinery of cscRNA biogenesis is unclear, but the cross-strand junction events show some features related to RNA splicing. The present study is a comprehensive survey of the non-canonical cross-strand RNA junction events, a resource for further characterization of the originations and functions of the cscRNAs.

scholarly journals Single molecule counting coupled to rapid amplification enables detection of αSynuclein aggregates in cerebrospinal fluid of PD patients

2021 ◽  
Author(s):  
Akshay BHUMKAR ◽  
Chloe MAGNAN ◽  
Derrick LAU ◽  
Eugene Soh Wei Jun ◽  
Nicolas DZAMKO ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Single Molecule ◽  
Rapid Amplification

scholarly journals Targeting BC200/miR218-5p Signaling Axis for Overcoming Temozolomide Resistance and Suppressing Glioma Stemness

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1859 ◽  
Author(s):  
Yu-Kai Su ◽  
Jia Wei Lin ◽  
Jing-Wen Shih ◽  
Hao-Yu Chuang ◽  
Iat-Hang Fong ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Bioinformatic Analysis ◽  
Benign Tumors ◽  
Rna Expression ◽  
Ki 67 ◽  
Normal Tissues ◽  
Significant Difference ◽  
Signaling Axis

Background: Glioblastoma (GB) is one of the most common (~30%) and lethal cancers of the central nervous system. Although new therapies are emerging, chemoresistance to treatment is one of the major challenges in cancer treatment. Brain cytoplasmic 200 (BC200) RNA, also known as BCYRN1, is a long noncoding RNA (lncRNA) that has recently emerged as one of the crucial members of the lncRNA family. BC200 atypical expression is observed in many human cancers. BC200 expression is higher in invasive cancers than in benign tumors. However, the clinical significance of BC200 and its effect on GB multiforme is still unexplored and remains unclear. Methods: BC200 expression in GB patients and cell lines were investigated through RT-qPCR, immunoblotting, and immunohistochemistry analysis. The biological importance of BC200 was investigated in vitro and in vivo through knockdown and overexpression. Bioinformatic analysis was performed to determine miRNAs associated with BC200 RNA. Results: Our findings revealed that in GB patients, BC200 RNA expression was higher in blood and tumor tissues than in normal tissues. BC200 RNA expression have a statistically significant difference between the IDH1 and P53 status. Moreover, the BC200 RNA expression was higher than both p53, a prognostic marker of glioma, and Ki-67, a reliable indicator of tumor cell proliferation activity. Overexpression and silencing of BC200 RNA both in vitro and in vivo significantly modulated the proliferation, self-renewal, pluripotency, and temozolomide (TMZ) chemo-resistance of GB cells. It was found that the expressions of BC200 were up-regulated and that of miR-218-5p were down-regulated in GB tissues and cells. miR-218-5p inhibited the expression of BC200. Conclusions: This study is the first to show that the molecular mechanism of BC200 promotes GB oncogenicity and TMZ resistance through miR-218-5p expression modulation. Thus, the noncoding RNA BC200/miR-218-5p signaling circuit is a potential clinical biomarker or therapeutic target for GB.

scholarly journals RAISING: a high-performance method for identifying random transgene integration sites

2021 ◽  
Author(s):  
Yusaku Wada ◽  
Tomoo Sato ◽  
Hiroo Hasegawa ◽  
Takahiro Matsudaira ◽  
Naganori Nao ◽  
...  
Keyword(s):  
High Performance ◽  
Viral Vectors ◽  
Viral Infections ◽  
Therapeutic Interventions ◽  
Transgene Integration ◽  
Adult T Cell Leukemia ◽  
Integration Sites ◽  
Infected Cells ◽  
Novel Method ◽  
Rapid Amplification

Abstract Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking transgene integration sites. RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) is a sensitive, inexpensive alternative to established methods. Its compatibility with Sanger sequencing combined with our CLOVA (Clonality Value) software is critical for those without access to expensive next-generation sequencing. To model our method, we analyzed samples from 698 patients infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL). We defined a clonality value identifying ATL patients with 100% sensitivity and 95.3% specificity, and our preliminary longitudinal analysis suggests this may also be useful for ATL risk assessment. We anticipate future studies will confirm the broad applicability of our technology, especially in the emerging gene therapy sector.

scholarly journals Structural mechanism of DNA-end synapsis in the non-homologous end joining pathway for repairing double-strand breaks: bridge over troubled ends

2019 ◽  
Vol 47 (6) ◽  
pp. 1609-1619 ◽  
Author(s):  
Qian Wu
Keyword(s):  
Single Molecule ◽  
Genome Instability ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Structural Mechanism ◽  
Strand Breaks ◽  
Single Molecule Studies ◽  
Non Homologous End Joining ◽  
Dna Ends

Non-homologous end joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), which is the most toxic DNA damage in cells. Unrepaired DSBs can cause genome instability, tumorigenesis or cell death. DNA end synapsis is the first and probably the most important step of the NHEJ pathway, aiming to bring two broken DNA ends close together and provide structural stability for end processing and ligation. This process is mediated through a group of NHEJ proteins forming higher-order complexes, to recognise and bridge two DNA ends. Spatial and temporal understanding of the structural mechanism of DNA-end synapsis has been largely advanced through recent structural and single-molecule studies of NHEJ proteins. This review focuses on core NHEJ proteins that mediate DNA end synapsis through their unique structures and interaction properties, as well as how they play roles as anchor and linker proteins during the process of ‘bridge over troubled ends'.

scholarly journals XLF acts as a flexible connector during non-homologous end joining

2020 ◽  
Author(s):  
Sean M. Carney ◽  
Andrew T. Moreno ◽  
Sadie C. Piatt ◽  
Metztli Cisneros-Aguirre ◽  
Felicia Wednesday Lopezcolorado ◽  
...  
Keyword(s):  
Single Molecule ◽  
Critical Factor ◽  
Tail Length ◽  
Binding Motif ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Synaptic Complex ◽  
Intrinsically Disordered ◽  
Non Homologous End Joining ◽  
Dna Ends

AbstractNon-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for end joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.

scholarly journals The Genome of the Anaerobic Fungus Orpinomyces sp. Strain C1A Reveals the Unique Evolutionary History of a Remarkable Plant Biomass Degrader

2013 ◽  
Vol 79 (15) ◽  
pp. 4620-4634 ◽  
Author(s):  
Noha H. Youssef ◽  
M. B. Couger ◽  
Christopher G. Struchtemeyer ◽  
Audra S. Liggenstoffer ◽  
Rolf A. Prade ◽  
...  
Keyword(s):  
Single Molecule ◽  
Crop Residues ◽  
Plant Biomass ◽  
Consolidated Bioprocessing ◽  
Genomic Analysis ◽  
Comparative Genomic ◽  
Evolutionary Trajectory ◽  
Content Type ◽  
Gene Acquisition ◽  
Intergenic Regions

ABSTRACTAnaerobic gut fungi represent a distinct early-branching fungal phylum (Neocallimastigomycota) and reside in the rumen, hindgut, and feces of ruminant and nonruminant herbivores. The genome of an anaerobic fungal isolate,Orpinomycessp. strain C1A, was sequenced using a combination of Illumina and PacBio single-molecule real-time (SMRT) technologies. The large genome (100.95 Mb, 16,347 genes) displayed extremely low G+C content (17.0%), large noncoding intergenic regions (73.1%), proliferation of microsatellite repeats (4.9%), and multiple gene duplications. Comparative genomic analysis identified multiple genes and pathways that are absent in Dikarya genomes but present in early-branching fungal lineages and/or nonfungal Opisthokonta. These included genes for posttranslational fucosylation, the production of specific intramembrane proteases and extracellular protease inhibitors, the formation of a complete axoneme and intraflagellar trafficking machinery, and a near-complete focal adhesion machinery. Analysis of the lignocellulolytic machinery in the C1A genome revealed an extremely rich repertoire, with evidence of horizontal gene acquisition from multiple bacterial lineages. Experimental analysis indicated that strain C1A is a remarkable biomass degrader, capable of simultaneous saccharification and fermentation of the cellulosic and hemicellulosic fractions in multiple untreated grasses and crop residues examined, with the process significantly enhanced by mild pretreatments. This capability, acquired during its separate evolutionary trajectory in the rumen, along with its resilience and invasiveness compared to prokaryotic anaerobes, renders anaerobic fungi promising agents for consolidated bioprocessing schemes in biofuels production.

DNA Aneuploidy in Benign Tumors and Normal Tissues of the Female Genital Tract

1996 ◽  
Vol 15 (1) ◽  
pp. 63-68 ◽  
Author(s):  
Chyong-Huey Lai ◽  
Swei Hsueh ◽  
Kuo-Chien Tsao ◽  
Hsiu-Mei Chen
Keyword(s):  
Genital Tract ◽  
Female Genital Tract ◽  
Benign Tumors ◽  
Dna Aneuploidy ◽  
Normal Tissues ◽  
Female Genital

scholarly journals Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair

2015 ◽  
Vol 112 (20) ◽  
pp. E2575-E2584 ◽  
Author(s):  
Dylan A. Reid ◽  
Sarah Keegan ◽  
Alejandra Leo-Macias ◽  
Go Watanabe ◽  
Natasha T. Strande ◽  
...  
Keyword(s):  
Single Molecule ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dna Ligase ◽  
End Joining ◽  
Dna Ligase Iv ◽  
Ligase Iv ◽  
End To End

Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.

scholarly journals The Fusion of CLEC12A and MIR223HG Arises from a trans-Splicing Event in Normal and Transformed Human Cells

2021 ◽  
Vol 22 (22) ◽  
pp. 12178
Author(s):  
Bijay P. Dhungel ◽  
Geoffray Monteuuis ◽  
Caroline Giardina ◽  
Mehdi S. Tabar ◽  
Yue Feng ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Cellular Localization ◽  
Chromosomal Rearrangements ◽  
Chimeric Protein ◽  
Cell Surface Receptor ◽  
Lectin Domain ◽  
Normal Tissues ◽  
Chimeric Rnas ◽  
Trans Splicing

Chimeric RNAs are often associated with chromosomal rearrangements in cancer. In addition, they are also widely detected in normal tissues, contributing to transcriptomic complexity. Despite their prevalence, little is known about the characteristics and functions of chimeric RNAs. Here, we examine the genetic structure and biological roles of CLEC12A-MIR223HG, a novel chimeric transcript produced by the fusion of the cell surface receptor CLEC12A and the miRNA-223 host gene (MIR223HG), first identified in chronic myeloid leukemia (CML) patients. Surprisingly, we observed that CLEC12A-MIR223HG is not just expressed in CML, but also in a variety of normal tissues and cell lines. CLEC12A-MIR223HG expression is elevated in pro-monocytic cells resistant to chemotherapy and during monocyte-to-macrophage differentiation. We observed that CLEC12A-MIR223HG is a product of trans-splicing rather than a chromosomal rearrangement and that transcriptional activation of CLEC12A with the CRISPR/Cas9 Synergistic Activation Mediator (SAM) system increases CLEC12A-MIR223HG expression. CLEC12A-MIR223HG translates into a chimeric protein, which largely resembles CLEC12A but harbours an altered C-type lectin domain altering key disulphide bonds. These alterations result in differences in post-translational modifications, cellular localization, and protein–protein interactions. Taken together, our observations support a possible involvement of CLEC12A-MIR223HG in the regulation of CLEC12A function. Our workflow also serves as a template to study other uncharacterized chimeric RNAs.

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