scholarly journals CDK13 RNA Over-Editing Mediated by ADAR1 Associates with Poor Prognosis of Hepatocellular Carcinoma Patients

2018 ◽  
Vol 47 (6) ◽  
pp. 2602-2612 ◽  
Author(s):  
Xiuqing Dong ◽  
Geng Chen ◽  
Zhixiong Cai ◽  
Zhenli Li ◽  
Liman Qiu ◽  
...  
Keyword(s):  
Rna Editing ◽  
Poor Prognosis ◽  
Tissue Sample ◽  
Rna Seq ◽  
Loss Of Function ◽  
Tissue Samples ◽  
Cancer Driver ◽  
Adenosine Deaminases ◽  
Depth Analysis ◽  
Aberrant Rna

Background/Aims: Aberrant RNA editing, mediated by adenosine deaminases acting on RNA (ADAR), serves as a post-transcriptional event participating in tumorigenesis and prognosis. However, the RNA editing profiles during HCC progression and their clinical correlations remain unclear. Methods: Multiple tissue samples were collected from an advanced HCC patient. RNA-seq was performed to obtain the RNA editing profiles for each sample. Two RNA editing sites from CDK13 were further validated in 60 HCC patients; and their potential regulatory mechanisms were investigated. Results: In-depth analysis of the RNA-seq data revealed a significant number of editing sites (632-816) in coding regions for each tissue sample, showing branched evolution during tumorigenesis and metastasis. Two editing sites (Q103R and K96R) in CDK13 showed significant over-editing in tumor, and these phenomenon were validated in 60 HCC patients. Furthermore, the clinicopathological analysis revealed that these CDK13 over-editing sites were positively associated with TNM, PVTT and poor prognosis. In addition, the editing level of these sites were significantly correlated with the expression of ADAR1. Loss of function assays further proved that these CDK13 over-editing sites were mediated by ADAR1 in HCC cells. Conclusions: CDK13 RNA over-editing sites mediated by ADAR1 may serve as novel cancer driver events in HCC progression.

scholarly journals Developmental atlas of the RNA editome in Sus scrofa skeletal muscle

DNA Research ◽  
2019 ◽  
Vol 26 (3) ◽  
pp. 261-272 ◽  
Author(s):  
Yalan Yang ◽  
Min Zhu ◽  
Xinhao Fan ◽  
Yilong Yao ◽  
Junyu Yan ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Rna Editing ◽  
Sus Scrofa ◽  
Muscle Development ◽  
Rna Seq ◽  
Sequencing Data ◽  
Adenosine Deaminases ◽  
Bisulphite Sequencing ◽  
Highly Correlated ◽  
Gain Loss

AbstractAdenosine-to-inosine (A-to-I) RNA editing meditated by adenosine deaminases acting on RNA (ADARs) enzymes is a widespread post-transcriptional event in mammals. However, A-to-I editing in skeletal muscle remains poorly understood. By integrating strand-specific RNA-seq, whole genome bisulphite sequencing, and genome sequencing data, we comprehensively profiled the A-to-I editome in developing skeletal muscles across 27 prenatal and postnatal stages in pig, an important farm animal and biomedical model. We detected 198,892 A-to-I editing sites and found that they occurred more frequently at prenatal stages and showed low conservation among pig, human, and mouse. Both the editing level and frequency decreased during development and were positively correlated with ADAR enzymes expression. The hyper-edited genes were functionally related to the cell cycle and cell division. A co-editing module associated with myogenesis was identified. The developmentally differential editing sites were functionally enriched in genes associated with muscle development, their editing levels were highly correlated with expression of their host mRNAs, and they potentially influenced the gain/loss of miRNA binding sites. Finally, we developed a database to visualize the Sus scrofa RNA editome. Our study presents the first profile of the dynamic A-to-I editome in developing animal skeletal muscle and provides evidences that RNA editing is a vital regulator of myogenesis.

scholarly journals Investigation of RNA Editing Sites within Bound Regions of RNA-Binding Proteins

2019 ◽  
Vol 8 (4) ◽  
pp. 19 ◽  
Author(s):  
Tyler Weirick ◽  
Giuseppe Militello ◽  
Mohammed Rabiul Hosen ◽  
David John ◽  
Joseph B. Moore ◽  
...  
Keyword(s):  
Rna Editing ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Seq ◽  
Rna Modifications ◽  
Adenosine Deaminases ◽  
Sequencing Errors ◽  
Additional Mode ◽  
Editing Enzyme

Studies in epitranscriptomics indicate that RNA is modified by a variety of enzymes. Among these RNA modifications, adenosine to inosine (A-to-I) RNA editing occurs frequently in the mammalian transcriptome. These RNA editing sites can be detected directly from RNA sequencing (RNA-seq) data by examining nucleotide changes from adenosine (A) to guanine (G), which substitutes for inosine (I). However, a careful investigation of such nucleotide changes must be conducted to distinguish sequencing errors and genomic mutations from the genuine editing sites. Building upon our recent introduction of an easy-to-use bioinformatics tool, RNA Editor, to detect RNA editing events from RNA-seq data, we examined the extent by which RNA editing events affect the binding of RNA-binding proteins (RBP). Through employing bioinformatic techniques, we uncovered that RNA editing sites occur frequently in RBP-bound regions. Moreover, the presence of RNA editing sites are more frequent when RNA editing islands were examined, which are regions in which RNA editing sites are present in clusters. When the binding of one RBP, human antigen R [HuR; encoded by ELAV-like protein 1 (ELAV1)], was quantified experimentally, its binding was reduced upon silencing of the RNA editing enzyme adenosine deaminases acting on RNA (ADAR) compared to the control—suggesting that the presence of RNA editing islands influence HuR binding to its target regions. These data indicate RNA editing as an important mediator of RBP–RNA interactions—a mechanism which likely constitutes an additional mode of post-transcription gene regulation in biological systems.

scholarly journals Loss of Asxl1 Cooperates with Oncogenic Nras to Drive CMML Progression

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3790-3790
Author(s):  
Xiaona You ◽  
Zhi Wen ◽  
Guangyao Kong ◽  
Adhithi Rajagopalan ◽  
Erik A. Ranheim ◽  
...  
Keyword(s):  
Median Survival ◽  
Poor Prognosis ◽  
Single Agent ◽  
Myeloproliferative Neoplasm ◽  
Erk Signaling ◽  
Rna Seq ◽  
Significant Group ◽  
Depth Analysis

Chronic myelomonocytic leukemia (CMML) belongs to the group of "mixed myelodysplastic/myeloproliferative neoplasm". There is currently no effective chemotherapy to treat CMML. Approximately 30% of CMML cases evolve to acute myeloid leukemia (AML) soon after their initial diagnosis, contributing to the poor prognosis of CMML patients. Ras pathway genes are frequently mutated in CMML patients (NRAS:11%; CBL:10%; KRAS:8%). Our whole exome sequencing of CMML patient samples and targeted sequencing of CMML patient-derived myeloid colonies showed that oncogenic NRAS could serve as an initiating or progression mutation. Consistent with human genetics, all the oncogenic Nras mice we and others characterized (NrasG12D/+, NrasG12D/G12D, and NrasQ61R/+), develop myeloproliferative (MP)-CMML like phenotypes.However, oncogenic NRAS alone is insufficient to drive CMML progression or transformation to AML. Additional sex combs-like 1 (ASXL1) is a human homolog of fly Asx. Mutations in ASXL1are predominantly nonsense and frequently identified in all myeloid malignancies (e.g. 40% in CMML). ASXL1mutations predict inferior overall survival in univariable analysis in multiple large CMML cohort studies and are significantly associated with NRASmutations in CMML patients. Therefore, we hypothesize that loss of Asxl1 cooperates with oncogenic Nrasto drive CMML progression. To test our hypothesis, we used Vav-Cre to drive oncogenic Nras expression and/or Asxl1 deletion in hematopoietic system. We refer these mice as NrasG12D/+, Asxl1-/-and NrasG12D/+;Asxl1-/-(NA) mice. Consistent with the literature, Asxl1-/-mice are normal at 6-week old. NrasG12D/+ mice showed mild CMML-like phenotypes, including enlarged spleen, increased white blood cell and monocyte counts, and expanded LSKs (Lin-Sca-1+c-Kit+) and myeloid progenitors (MPs). Compared to NrasG12D/+ mice, NA mice showed more sever phenotypes that were associated with hyperactivated ERK signaling in MPs at both basal level and upon GM-CSF stimulation. Our new NrasG12D/+mice developed a fully penetrant MP-CMML with the median survival of ~430 days. Loss of Asxl1 significantly shortened the median survival of NrasG12D/+ mice to 220 days. Half of NA mice displayed CMML transformation to AML. The transformed AML was serially transplantable to recipients. To investigate the mechanism(s) underlying NA-driven CMML/AML, we performed RNA-Seq analysis in Lin- c-Kit+BM cells from age-matched control, Asxl1-/-, NrasG12D/+, and moribund NA mice with CMML or AML phenotypes. We found 844 differentially expressed genes (DEG) in AML cells compared to control cells (FDR<0.05 and fold change>2). Among these genes, 341 are common between NA and published Nf1+/-;Asxl1+/-AML cells, including AP-1 complex genes (Jun, Junb, Jund and Fosb) that are crucial for malignant propagation in several AML subtypes. The remaining 503 genes are unique in NA AML, including Flt3, one of the mostly mutated genes in AML. These data suggest that our NA AML model demonstrates some distinct mechanisms from Nf1+/-;Asxl1+/-model. In-depth analysis of RNA-seq data is still ongoing. We also examined multiple histone marks using Western blot in BM cells of 6-week old mice. Our data showed that levels of H3K4me3, H3K4me1, and H3K27me3 were downregulated in Asxl1-/-cells, whereas H3K27Ac level was comparable to that of control cells. In contrast, H3K27Ac level was upregulated in NrasG12D/+and NA cells, while levels of H3K4me3, H3K4me1, and H3K27me3 were indistinguishable from those of control cells. BRD4 is a member of the BET family of bromodomain-containing proteins that binds to acetylated histones to activate gene transcription. Thus, we hypothesized that Ras/MEK/ERK signaling and BRD/BET are potential targets to inhibit AML progression. To test this hypothesis, we treated the AML cells and BMT recipients using trametinib (a MEK inhibitor approved by FDA to treat melanoma) and GSK525762 (a pan BET inhibitor under clinical development). We demonstrated thatcombined treatment significantly inhibited the growth of leukemia cells in vitro and prolonged the survival of recipients in vivo. It was more effective than single agent alone. Overall, we established a novel CMML/AML mouse model, which represents a significant group of CMML patients with poor prognosis. Our study provides a strong rational to treat these patients with combined MEK and BET inhibition. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Determining the cellular localization of C.elegans Editing Enzyme

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Anna Dudley ◽  
Heather Hundley ◽  
Suba Rajendren
Keyword(s):  
Rna Editing ◽  
Cellular Localization ◽  
Nuclear Retention ◽  
Western Blots ◽  
Adenosine Deaminases ◽  
C Elegans ◽  
Sds Page ◽  
Aberrant Rna ◽  
Editing Enzyme ◽  
Adar Editing

Background and Hypothesis:  Over two thirds of human mRNAs contain adenosine(A)-to-inosine (I) editing sites indicating that RNA editing significantly alters the flow of genetic information. RNA editing is required for normal development and proper neuronal function in all animals. Aberrant RNA editing is identified in several neurological disorders and cancers.   A-to-I RNA editing is catalyzed by adenosine deaminases that act on RNA (ADARs) proteins. These enzymes commonly bind to double stranded RNA (dsRNA) and catalyze the conversion of adenosine to inosine. However, it is unknown whether these different regions of mRNA are edited by ADARs in the cytoplasm or nucleus. Early research has shown that inosines are present in the nucleus, while new additional evidence shows activity in cytoplasm as well. What dictates the location of the editing is yet unknown.   Experimental Design:   The subcellular localization of the ADAR editing enzyme will be determined by performing western blots of fractionated Caenorhabditis elegans embryos. Unlike mammals, ADAR editing is not essential in C. elegans, thus making it an easy system to address mechanistic questions about RNA editing. Embryos will be attained from wild-type, ADR-1 and ADR-2 knockout worms, and biochemical techniques will be used to obtain nuclei and cytoplasmic fractions. These fractions will be subjected to SDS-PAGE and western blotting for the ADAR editing enzyme, ADR-2. I will also use positive controls of a nuclear protein (histone) and a cytoplasmic protein (Tubulin) to test the fractionation.  Anticipated Results:   That there will be more ADR-2 enzyme in the nucleus than the cytoplasm.  Potential Impact:  Knowing the location of the edited RNA will allow researchers to have a better idea of what mechanisms influence the editing of ADAR, and what dictates the localization of dsRNA. Current theories involve the number of inosine groups, cellular conditions which effect localization, and nuclear retention molecules other than inosine. 

Biomedical applications: Pitfalls in the practice

1994 ◽  
Vol 52 ◽  
pp. 378-379
Author(s):  
J. D. Shelburne ◽  
Peter Ingram ◽  
Victor L. Roggli ◽  
Ann LeFurgey
Keyword(s):  
Operating Room ◽  
Liquid Nitrogen ◽  
Lung Tissue ◽  
Biomedical Applications ◽  
Microprobe Analysis ◽  
Tissue Sample ◽  
Cellular Level ◽  
Tissue Samples ◽  
Asbestos Fibers

At present most medical microprobe analysis is conducted on insoluble particulates such as asbestos fibers in lung tissue. Cryotechniques are not necessary for this type of specimen. Insoluble particulates can be processed conventionally. Nevertheless, it is important to emphasize that conventional processing is unacceptable for specimens in which electrolyte distributions in tissues are sought. It is necessary to flash-freeze in order to preserve the integrity of electrolyte distributions at the subcellular and cellular level. Ideally, biopsies should be flash-frozen in the operating room rather than being frozen several minutes later in a histology laboratory. Electrolytes will move during such a long delay. While flammable cryogens such as propane obviously cannot be used in an operating room, liquid nitrogen-cooled slam-freezing devices or guns may be permitted, and are the best way to achieve an artifact-free, accurate tissue sample which truly reflects the in vivo state. Unfortunately, the importance of cryofixation is often not understood. Investigators bring tissue samples fixed in glutaraldehyde to a microprobe laboratory with a request for microprobe analysis for electrolytes.

Artificial RNA Editing with ADAR for Gene Therapy

2020 ◽  
Vol 20 (1) ◽  
pp. 44-54 ◽  
Author(s):  
Sonali Bhakta ◽  
Toshifumi Tsukahara
Keyword(s):  
Gene Therapy ◽  
Genetic Code ◽  
Molecular Mechanism ◽  
Rna Editing ◽  
Comparative Studies ◽  
Genetic Diseases ◽  
Adenosine Deaminases ◽  
Family Protein ◽  
Hydrolytic Deamination

Editing mutated genes is a potential way for the treatment of genetic diseases. G-to-A mutations are common in mammals and can be treated by adenosine-to-inosine (A-to-I) editing, a type of substitutional RNA editing. The molecular mechanism of A-to-I editing involves the hydrolytic deamination of adenosine to an inosine base; this reaction is mediated by RNA-specific deaminases, adenosine deaminases acting on RNA (ADARs), family protein. Here, we review recent findings regarding the application of ADARs to restoring the genetic code along with different approaches involved in the process of artificial RNA editing by ADAR. We have also addressed comparative studies of various isoforms of ADARs. Therefore, we will try to provide a detailed overview of the artificial RNA editing and the role of ADAR with a focus on the enzymatic site directed A-to-I editing.

scholarly journals Differential Expression of BARD1 Isoforms in Melanoma

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 320
Author(s):  
Lorissa I. McDougall ◽  
Ryan M. Powell ◽  
Magdalena Ratajska ◽  
Chi F. Lynch-Sutherland ◽  
Sultana Mehbuba Hossain ◽  
...  
Keyword(s):  
Long Range ◽  
Patient Outcomes ◽  
Splice Variants ◽  
Significant Proportion ◽  
Nanopore Sequencing ◽  
Rna Seq ◽  
Splice Isoforms ◽  
Tissue Samples ◽  
Multiple Transcripts ◽  
Mrna Variants

Melanoma comprises <5% of cutaneous malignancies, yet it causes a significant proportion of skin cancer-related deaths worldwide. While new therapies for melanoma have been developed, not all patients respond well. Thus, further research is required to better predict patient outcomes. Using long-range nanopore sequencing, RT-qPCR, and RNA sequencing analyses, we examined the transcription of BARD1 splice isoforms in melanoma cell lines and patient tissue samples. Seventy-six BARD1 mRNA variants were identified in total, with several previously characterised isoforms (γ, φ, δ, ε, and η) contributing to a large proportion of the expressed transcripts. In addition, we identified four novel splice events, namely, Δ(E3_E9), ▼(i8), IVS10+131▼46, and IVS10▼176, occurring in various combinations in multiple transcripts. We found that short-read RNA-Seq analyses were limited in their ability to predict isoforms containing multiple non-contiguous splicing events, as compared to long-range nanopore sequencing. These studies suggest that further investigations into the functional significance of the identified BARD1 splice variants in melanoma are warranted.

scholarly journals The Dutch National TissueArchive Portal enables efficient, consistent, and transparent procurement of diagnostic tissue samples for scientific use

2021 ◽  
Author(s):  
Robin Verjans ◽  
Annette H. Bruggink ◽  
Robby Kibbelaar ◽  
Jos Bart ◽  
Aletta Debernardi ◽  
...  
Keyword(s):  
The Netherlands ◽  
Tissue Sample ◽  
Ffpe Tissue ◽  
Tissue Samples ◽  
Internet Portal ◽  
Formalin Fixed Paraffin ◽  
Practical Support ◽  
Formalin Fixed Paraffin Embedded ◽  
The Rich ◽  
Formalin Fixed

AbstractBiobanks play a crucial role in enabling biomedical research by facilitating scientific use of valuable human biomaterials. The PALGA foundation—a nationwide network and registry of histo- and cytopathology in the Netherlands—was established to promote the provision of data within and between pathology departments, and to make the resulting knowledge available for healthcare. Apart from the pathology data, we aimed to utilize PALGA’s nationwide network to find and access the rich wealth of Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples for scientific use.  We implemented the Dutch National TissueArchive Portal (DNTP) to utilize PALGA’s nationwide network for requesting FFPE tissue samples. The DNTP consists of (1) a centrally organized internet portal to improve the assessing, processing, harmonization, and monitoring of the procurement process, while (2) dedicated HUB-employees provide practical support at peripheral pathology departments. Since incorporation of the DNTP, both the number of filed requests for FFPE tissue samples and the amount of HUB-mediated support increased 55 and 29% respectively. In line, the sample procurement duration time decreased significantly (− 47%). These findings indicate that implementation of the DNTP improved the frequency, efficiency, and transparency of FFPE tissue sample procurement for research in the Netherlands. To conclude, the need for biological resources is growing persistently to enable precision medicine. Here, we access PALGA’s national, pathology network by implementation of the DNTP to allow for efficient, consistent, and transparent exchange of FFPE tissue samples for research across the Netherlands.

scholarly journals MBRS-17. EXAMINING THE ROLE OF LHX9 IN GROUP 3 MEDULLOBLASTOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii401-iii401
Author(s):  
Sarah Injac ◽  
L Frank Huang ◽  
Stephen Mack ◽  
Frank Braun ◽  
Yuchen Du ◽  
...  
Keyword(s):  
Drug Repositioning ◽  
Single Agent ◽  
Xenograft Model ◽  
Cell Killing ◽  
Rna Seq ◽  
Loss Of Function ◽  
Novel Treatments ◽  
Novel Approach ◽  
Group 3

Abstract Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Despite major advances in our understanding of the biology of MB, novel treatments remain urgently needed. Using a chemical-genomics driven drug repositioning strategy, we identified the cardiac glycoside family of compounds as potential treatments for Group 3 MB. We subsequently demonstrated that single-agent treatment with digoxin prolongs survival in a patient-derived xenograft model (PDOX) of Group 3 MB to a degree comparable to radiation therapy, a mainstay in the treatment of MB. Finally, we examined the mechanism of digoxin-mediated cell killing using RNA-seq. This work identified LHX9, a member of the LIM homeobox family of transcription factors, as the gene most significantly down-regulated following treatment (Huang and Injac et al, Sci Trans Medicine, 2018). Homologs of LHX9 play key roles in cerebellar development via spatially and temporally restricted expression and LHX9 has been proposed as a core transcription factor (TF) in the regulatory circuitry of Group 3 tumors. Loss of function of other core TFs has been shown to impact MB growth. The role of LHX9 in MB, however, has not been previously experimentally evaluated. We now report that knockdown of LHX9 in MB-derived cell lines results in marked growth inhibition raising the possibility that loss of LHX9 plays a major role in digoxin-mediated cell killing and that LHX9 represents a key dependency required for the growth of Group 3 MB. Clinical targeting of core TFs would represent a novel approach to targeting this devastating disease.

A study on the genomic alterations of transformation from colorectal adenoma to colorectal cancer.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e15607-e15607
Author(s):  
Qingjian Chen ◽  
Pan Yang ◽  
Linna Luo ◽  
Wenhua Fan ◽  
Chen Wei ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cancer Patients ◽  
Tissue Sample ◽  
Intraepithelial Neoplasia ◽  
Stage I ◽  
Tissue Samples ◽  
Diagnostic Potential ◽  
Notch Pathways ◽  
Colorectal Cancer Patients ◽  
Villous Adenomas

e15607 Background: Colorectal cancer is one of the most common malignancies worldwide. Approximately 85% of colorectal cancers are thought to result from adenoma. However, the molecular mechanism of adenoma transformation into colorectal cancer is still unclear. Methods: Ninety-nine adenoma patients aged from 25 to 78 years old were enrolled in this study. We collected tissue sample from each patient and 77 matched blood samples. Pathological subtypes included tubular villous adenomas, villous adenomas, tubular adenomas, high-grade intraepithelial neoplasia, and polyps. Eighty-one stage I colorectal cancer patients (CRC I) were also enrolled in this study. All samples underwent Next-generation sequencing with a panel of 405 cancer related genes. Results: Mutational profiles of adenoma and CRC I patients were compared. The top 5 most frequently mutated genes in adenoma were APC (71%), KRAS (41%), ATM (33%), RIF1 (31%), SYNE1 (28%). While in CRC I patients, top 5 mutated genes were APC (78%), TP53 (57%), TTN (35%), KRAS (33%) and TCF7L2 (22%). There were significant differences between TP53 and TTN by chi-square test. The frequency, number and TMB of mutations in stage I colorectal cancer patients were significantly higher than those in various adenoma subtypes. Stage I colorectal cancer patients have more mutated genes enriched in the Wnt and Notch pathways than adenoma patients. We analyzed mutation signatures in CRC I and adenoma patients, and CRC I were more focused on mutation signatures of mismatch repair such as signature 1, signature 6, signature 10, and signature 15. A total of 391 mutations were identified in tissue samples, while 130 mutations were found in plasma cell-free DNA, with 116 mutations shared between them. The two genes with the highest consistency between tissue and blood were PAX7 and KMT2D. Conclusions: TP53 and TTN are associated with the transition from CRC I to adenoma, and Wnt and Notch pathways may also be involved. PAX7 and KMT2D mutations frequently found in adenoma tissue and blood cfDNA demonstrate the diagnostic potential of these two genes in clinic.

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