ORCID Profile
0000-0002-8663-4586
Current Organisations
Centre for Genomic Regulation
,
University of New South Wales
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Publisher: American Association for Cancer Research (AACR)
Date: 07-2018
DOI: 10.1158/1538-7445.AM2018-2360
Abstract: Certain aspects of diagnosis, prognosis, and treatment of cancer patients are still important challenges to be addressed. We developed a pipeline to uncover patterns of alternative polyadenylation (APA), a hidden complexity in cancer transcriptomes, to further accelerate efforts to discover novel cancer genes and pathways. Here, we found a significant shift in usage of poly(A) signals in six common tumor types compared to normal tissues. We further defined specific subsets of APA events to efficiently classify cancer types/subtypes. Triple negative breast cancers, for ex le, have specific 3'UTR length alterations where the significant majority are shortening events (70%, 113 of 165) of mostly proliferation-related transcripts compared with normal breast tissue. Such shortening events correlate with increased protein levels and relapse free survival of patients, suggesting functional significance of isoform variability. In line with this isoform ersity, we also detected deregulated expression of mRNA polyadenylation complex proteins in breast cancer cells. Of note, APA proteins are responsive to proliferative signals including estrogen and epidermal growth factor, suggesting a potential explanation to 3'-end isoform ersity in cancer cells. Overall, our study offers a computational and experimental approach for use of APA in novel gene discovery and classification in common tumor types, with important implications in basic research, biomarker discovery, and precision medicine approaches. Citation Format: Oguzhan Begik, Melda Ercan, Harun Cingoz, Tolga Can, Merve Oyken, Ayse Elif Erson-Bensan. Deregulated APA and cancer specific APA isoforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018 2018 Apr 14-18 Chicago, IL. Philadelphia (PA): AACR Cancer Res 2018 (13 Suppl):Abstract nr 2360.
Publisher: Cold Spring Harbor Laboratory
Date: 04-12-2019
DOI: 10.1101/864322
Abstract: Nanopore sequencing has enabled sequencing of native RNA molecules without conversion to cDNA, thus opening the gates to a new era for the unbiased study of RNA biology. However, a formal barcoding protocol for direct sequencing of native RNA molecules is currently lacking, limiting the efficient processing of multiple s les in the same flowcell. A major limitation for the development of barcoding protocols for direct RNA sequencing is the error rate introduced during the base-calling process, especially towards the 5’ and 3’ ends of reads, which complicates sequence-based barcode demultiplexing. Here, we propose a novel strategy to barcode and demultiplex direct RNA sequencing nanopore data, which does not rely on base-calling or additional library preparation steps. Specifically, custom DNA oligonucleotides are ligated to RNA transcripts during library preparation. Then, raw current signal corresponding to the DNA barcode is extracted and transformed into an array of pixels, which is used to determine the underlying barcode using a deep convolutional neural network classifier. Our method, DeePlexiCon , implements a 20-layer residual neural network model that can demultiplex 93% of the reads with 95.1% specificity, or 60% of reads with 99.9% specificity. The availability of an efficient and simple barcoding strategy for native RNA sequencing will enhance the use of direct RNA sequencing by making it more cost-effective to the entire community. Moreover, it will facilitate the applicability of direct RNA sequencing to s les where the RNA amounts are limited, such as patient-derived s les.
Publisher: Springer Science and Business Media LLC
Date: 19-12-2022
DOI: 10.1038/S41592-022-01714-W
Abstract: RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3′ end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3′ end, regardless of its polyadenylation status, without the need for PCR lification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide ersity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in in idual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.
Publisher: Cold Spring Harbor Laboratory
Date: 05-11-2019
DOI: 10.1101/830968
Abstract: RNA modifications play central roles in cellular fate and differentiation. These features have placed the epitranscriptome in the forefront of developmental biology and cancer research. However, the machinery responsible for placing, removing and recognizing more than 170 RNA modifications remains largely uncharacterized and poorly annotated, and we currently lack integrative studies that identify which RNA modification–related proteins (RMPs) may be dysregulated in each cancer type. Here we have performed a comprehensive annotation and evolutionary analysis of human RMPs as well as an integrative analysis of their expression patterns across 32 tissues, 10 species and 13,358 paired tumor-normal human s les. Our analysis reveals an unanticipated heterogeneity of RMP expression patterns across mammalian tissues, with a vast proportion of duplicated enzymes displaying testis-specific expression, suggesting a key role for RNA modifications in sperm formation and possibly intergenerational inheritance. Moreover, through the analysis of paired tumor-normal human s les we uncover many RMPs that are dysregulated in various types of cancer, and whose expression levels are predictive of cancer progression. Surprisingly, we find that several commonly studied RNA modification enzymes such as METTL3 or FTO, are not significantly up-regulated in most cancer types, once the s le is properly scaled and normalized to the full dataset, whereas several less-characterized RMPs, such as LAGE3 and HENMT1, are dysregulated in many cancers. Our analyses reveal an unanticipated heterogeneity in the expression patterns of RMPs across mammalian tissues, and uncover a large proportion of dysregulated RMPs in multiple cancer types. We provide novel targets for future cancer research studies targeting the human epitranscriptome, as well as foundations to understand cell type-specific behaviours that are orchestrated by RNA modifications.
Publisher: Cold Spring Harbor Laboratory
Date: 06-07-2020
DOI: 10.1101/2020.07.06.189969
Abstract: A broad ersity of modifications decorate RNA molecules. Originally conceived as static components, evidence is accumulating that some RNA modifications may be dynamic, contributing to cellular responses to external signals and environmental circumstances. A major difficulty in studying these modifications, however, is the need of tailored protocols to map each modification type in idually. Here, we present a new approach that uses direct RNA nanopore sequencing to identify and quantify RNA modifications present in native RNA molecules. First, we show that each RNA modification type results in a distinct and characteristic base-calling ‘error’ signature, which we validate using a battery of genetic strains lacking either pseudouridine (Y) or 2’-O-methylation (Nm) modifications. We then demonstrate the value of these signatures for de novo prediction of Y modifications transcriptome-wide, confirming known Y-modified sites as well as uncovering novel Y sites in mRNAs, ncRNAs and rRNAs, including a previously unreported Pus4-dependent Y modification in yeast mitochondrial rRNA, which we validate using orthogonal methods. To explore the dynamics of pseudouridylation across environmental stresses, we treat the cells with oxidative, cold and heat stresses, finding that yeast ribosomal rRNA modifications do not change upon environmental exposures, contrary to the general belief. By contrast, our method reveals many novel heat-sensitive Y-modified sites in snRNAs, snoRNAs and mRNAs, in addition to recovering previously reported sites. Finally, we develop a novel software, nanoRMS , which we show can estimate per-site modification stoichiometries from in idual RNA molecules by identifying the reads with altered current intensity and trace profiles, and quantify the RNA modification stoichiometry changes between two conditions. Our work demonstrates that Y RNA modifications can be predicted de novo and in a quantitative manner using native RNA nanopore sequencing.
Publisher: Informa UK Limited
Date: 02-03-2015
Publisher: Springer Science and Business Media LLC
Date: 09-09-2019
DOI: 10.1038/S41467-019-11713-9
Abstract: The epitranscriptomics field has undergone an enormous expansion in the last few years however, a major limitation is the lack of generic methods to map RNA modifications transcriptome-wide. Here, we show that using direct RNA sequencing, N 6 -methyladenosine (m 6 A) RNA modifications can be detected with high accuracy, in the form of systematic errors and decreased base-calling qualities. Specifically, we find that our algorithm, trained with m 6 A-modified and unmodified synthetic sequences, can predict m 6 A RNA modifications with ~90% accuracy. We then extend our findings to yeast data sets, finding that our method can identify m 6 A RNA modifications in vivo with an accuracy of 87%. Moreover, we further validate our method by showing that these ‘errors’ are typically not observed in yeast ime4 -knockout strains, which lack m 6 A modifications. Our results open avenues to investigate the biological roles of RNA modifications in their native RNA context.
Publisher: Cold Spring Harbor Laboratory
Date: 13-10-2023
Publisher: Springer Science and Business Media LLC
Date: 07-05-2020
DOI: 10.1186/S13059-020-02009-Z
Abstract: RNA modifications play central roles in cellular fate and differentiation. However, the machinery responsible for placing, removing, and recognizing more than 170 RNA modifications remains largely uncharacterized and poorly annotated, and we currently lack integrative studies that identify which RNA modification-related proteins (RMPs) may be dysregulated in each cancer type. Here, we perform a comprehensive annotation and evolutionary analysis of human RMPs, as well as an integrative analysis of their expression patterns across 32 tissues, 10 species, and 13,358 paired tumor-normal human s les. Our analysis reveals an unanticipated heterogeneity of RMP expression patterns across mammalian tissues, with a vast proportion of duplicated enzymes displaying testis-specific expression, suggesting a key role for RNA modifications in sperm formation and possibly intergenerational inheritance. We uncover many RMPs that are dysregulated in various types of cancer, and whose expression levels are predictive of cancer progression. Surprisingly, we find that several commonly studied RNA modification enzymes such as METTL3 or FTO are not significantly upregulated in most cancer types, whereas several less-characterized RMPs, such as LAGE3 and HENMT1, are dysregulated in many cancers. Our analyses reveal an unanticipated heterogeneity in the expression patterns of RMPs across mammalian tissues and uncover a large proportion of dysregulated RMPs in multiple cancer types. We provide novel targets for future cancer research studies targeting the human epitranscriptome, as well as foundations to understand cell type-specific behaviors that are orchestrated by RNA modifications.
Publisher: Cold Spring Harbor Laboratory
Date: 21-03-2023
DOI: 10.1101/2023.03.21.533606
Abstract: The biological relevance and dynamics of mRNA modifications have been extensively studied in the past few years, revealing their key roles in major cellular processes, such as cellular differentiation or sex determination. However, whether rRNA modifications are dynamically regulated, and under which conditions, remains largely unclear. Here, we performed a systematic characterization of bacterial rRNA modification dynamics upon exposure to erse antibiotics using native RNA nanopore sequencing. To identify significant rRNA modification changes, we developed NanoConsensus , a novel pipeline that integrates the estimates from multiple RNA modification detection algorithms, predicting differentially modified rRNA sites with very low false positive rates and high replicability. We showed that NanoConsensus is robust across RNA modification types, stoichiometries and coverage, and outperforms all in idual algorithms tested. Using this approach, we identified multiple rRNA modifications that are lost upon the presence of antibiotics, showing that rRNA modification profiles are altered in an antibiotic-specific manner. We found that significantly altered rRNA modified sites upon antibiotic exposure are located in the vicinity of the A and P-sites of the ribosome, possibly contributing to antibiotic resistance. We then systematically examined whether loss of ‘antibiotic-sensitive’ rRNA modifications may be sufficient to confer antibiotic resistance, finding that depletion of some rRNA modification enzymes guiding dysregulated rRNA modifications confers increased antibiotic resistance. Altogether, our work reveals that rRNA modification profiles can be rapidly altered in response to environmental exposures, and that nanopore sequencing can accurately identify dysregulated rRNA modifications, contributing to the mechanistic dissection of antibiotic resistance. Moreover, we provide a novel, robust workflow to study rRNA modification dynamics in any species using nanopore sequencing in a scalable and reproducible manner.
Publisher: Bentham Science Publishers Ltd.
Date: 16-08-2017
DOI: 10.2174/2211536606666170511102219
Abstract: RNA is chemically modified with over 100 distinct reactions. Among these reactions, methylation is probably the most extensively studied modification on the RNA molecule. Studies suggest methylation of Adenine residues (m6A) to be widespread in the transcriptome with potentially important roles in biological processes. Here, we review recent literature on m6A modification and potential implications for microRNA (miRNA) mediated gene expression regulation. These implications involve miRNA biogenesis, mRNA-miRNA interactions and m6A target selection. Understanding the extent and functions of m6A is likely to improve our understanding of the complexities of the transcriptome regulation in normal and in disease states.
Publisher: Springer Science and Business Media LLC
Date: 13-05-2021
DOI: 10.1038/S41587-021-00915-6
Abstract: Nanopore RNA sequencing shows promise as a method for discriminating and identifying different RNA modifications in native RNA. Expanding on the ability of nanopore sequencing to detect N
Publisher: Cold Spring Harbor Laboratory
Date: 14-09-2022
Abstract: Chemical RNA modifications, collectively referred to as the “epitranscriptome,” are essential players in fine-tuning gene expression. Our ability to analyze RNA modifications has improved rapidly in recent years, largely due to the advent of high-throughput sequencing methodologies, which typically consist of coupling modification-specific reagents, such as antibodies or enzymes, to next-generation sequencing. Recently, it also became possible to map RNA modifications directly by sequencing native RNAs using nanopore technologies, which has been applied for the detection of a number of RNA modifications, such as N6-methyladenosine (m 6 A), pseudouridine (Ψ), and inosine (I). However, the signal modulations caused by most RNA modifications are yet to be determined. A global effort is needed to determine the signatures of the full range of RNA modifications to avoid the technical biases that have so far limited our understanding of the epitranscriptome.
Publisher: IOP Publishing
Date: 26-04-2016
Publisher: Cold Spring Harbor Laboratory
Date: 21-01-2019
DOI: 10.1101/525741
Abstract: The field of epitranscriptomics has undergone an enormous expansion in the last few years however, a major limitation is the lack of generic methods to map RNA modifications transcriptome-wide. Here we show that using Oxford Nanopore Technologies, N6-methyladenosine (m6A) RNA modifications can be detected with high accuracy, in the form of systematic errors and decreased base-calling qualities. Our results open new avenues to investigate the universe of RNA modifications with single nucleotide resolution, in in idual RNA molecules.
Publisher: Cold Spring Harbor Laboratory
Date: 22-09-2021
DOI: 10.1101/2021.09.22.461331
Abstract: RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here, we develop Nanopore 3’ end-capture sequencing (Nano3P-seq), a novel method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition and tail length dynamics at per-read resolution. By employing a template switching-based sequencing protocol, Nano3P-seq can sequence any given RNA molecule from its 3’ end, regardless of its polyadenylation status, without the need for PCR lification or ligation of RNA adapters. We demonstrate that Nano3P-seq captures a wide ersity of RNA biotypes, providing quantitative estimates of RNA abundance and tail lengths in mRNA, lncRNA, sn/snoRNA, scaRNA, and rRNA molecules. We find that, in addition to mRNA and lncRNA, polyA tails can be identified in 16S mitochondrial rRNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we identify non-A bases within polyA tails of various lengths and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in in idual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.
Publisher: Elsevier BV
Date: 07-2017
No related grants have been discovered for Oguzhan Begik.