11 results on '"Oscar G. Wilkins"'
Search Results
2. Ultraplex: A rapid, flexible, all-in-one fastq demultiplexer [version 1; peer review: 2 approved]
- Author
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Oscar G Wilkins, Charlotte Capitanchik, Nicholas M. Luscombe, and Jernej Ule
- Subjects
Medicine ,Science - Abstract
Background: The first step of virtually all next generation sequencing analysis involves the splitting of the raw sequencing data into separate files using sample-specific barcodes, a process known as “demultiplexing”. However, we found that existing software for this purpose was either too inflexible or too computationally intensive for fast, streamlined processing of raw, single end fastq files containing combinatorial barcodes. Results: Here, we introduce a fast and uniquely flexible demultiplexer, named Ultraplex, which splits a raw FASTQ file containing barcodes either at a single end or at both 5’ and 3’ ends of reads, trims the sequencing adaptors and low-quality bases, and moves unique molecular identifiers (UMIs) into the read header, allowing subsequent removal of PCR duplicates. Ultraplex is able to perform such single or combinatorial demultiplexing on both single- and paired-end sequencing data, and can process an entire Illumina HiSeq lane, consisting of nearly 500 million reads, in less than 20 minutes. Conclusions: Ultraplex greatly reduces computational burden and pipeline complexity for the demultiplexing of complex sequencing libraries, such as those produced by various CLIP and ribosome profiling protocols, and is also very user friendly, enabling streamlined, robust data processing. Ultraplex is available on PyPi and Conda and via Github.
- Published
- 2021
- Full Text
- View/download PDF
3. Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD
- Author
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Sahba Seddighi, Yue A. Qi, Anna-Leigh Brown, Oscar G. Wilkins, Colleen Bereda, Cedric Belair, Yongjie Zhang, Mercedes Prudencio, Matthew J Keuss, Aditya Khandeshi, Sarah Pickles, Sarah E. Hill, James Hawrot, Daniel M. Ramos, Hebao Yuan, Jessica Roberts, Erika Kelmer Sacramento, Syed I. Shah, Mike A. Nalls, Jenn Colon-Mercado, Joel F. Reyes, Veronica H. Ryan, Matthew P. Nelson, Casey Cook, Ziyi Li, Laurel Screven, Justin Y Kwan, Anantharaman Shantaraman, Lingyan Ping, Yuka Koike, Björn Oskarsson, Nathan Staff, Duc M. Duong, Aisha Ahmed, Maria Secrier, Jerneg Ule, Steven Jacobson, Jonathan Rohrer, Andrea Malaspina, Jonathan D. Glass, Alessandro Ori, Nicholas T. Seyfried, Manolis Maragkakis, Leonard Petrucelli, Pietro Fratta, and Michael E. Ward
- Subjects
Article - Abstract
Functional loss of TDP-43, an RNA-binding protein genetically and pathologically linked to ALS and FTD, leads to inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. However, the possibility ofde novoprotein synthesis from cryptic exon transcripts has not been explored. Here, we show that mRNA transcripts harboring cryptic exons generatede novoproteins both in TDP-43 deficient cellular models and in disease. Using coordinated transcriptomic and proteomic studies of TDP-43 depleted iPSC-derived neurons, we identified numerous peptides that mapped to cryptic exons. Cryptic exons identified in iPSC models were highly predictive of cryptic exons expressed in brains of patients with TDP-43 proteinopathy, including cryptic transcripts that generatedde novoproteins. We discovered that inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Finally, we showed that thesede novopeptides were present in CSF from patients with ALS. The demonstration of cryptic exon translation suggests new mechanisms for ALS pathophysiology downstream of TDP-43 dysfunction and may provide a strategy for novel biomarker development.One Sentence SummaryLoss of TDP-43 function results in the expression ofde novoproteins from mis-spliced mRNA transcripts.
- Published
- 2023
4. Identifying ribosome heterogeneity using ribosome profiling
- Author
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Ferhat Alkan, Oscar G Wilkins, Santiago Hernández-Pérez, Sofia Ramalho, Joana Silva, Jernej Ule, and William J Faller
- Subjects
Ribosomal Proteins ,FOS: Clinical medicine ,Stem Cells ,Neurosciences ,Gene Expression ,Biochemistry & Proteomics ,Ecology,Evolution & Ethology ,RNA, Ribosomal ,Genetics ,Humans ,RNA, Messenger ,Ribosomes ,Genetics & Genomics ,Computational & Systems Biology - Abstract
Recent studies have revealed multiple mechanisms that can lead to heterogeneity in ribosomal composition. This heterogeneity can lead to preferential translation of specific panels of mRNAs, and is defined in large part by the ribosomal protein (RP) content, amongst other things. However, it is currently unknown to what extent ribosomal composition is heterogeneous across tissues, which is compounded by a lack of tools available to study it. Here we present dripARF, a method for detecting differential RP incorporation into the ribosome using Ribosome Profiling (Ribo-seq) data. We combine the ‘waste’ rRNA fragment data generated in Ribo-seq with the known 3D structure of the human ribosome to predict differences in the composition of ribosomes in the material being studied. We have validated this approach using publicly available data, and have revealed a potential role for eS25/RPS25 in development. Our results indicate that ribosome heterogeneity can be detected in Ribo-seq data, providing a new method to study this phenomenon. Furthermore, with dripARF, previously published Ribo-seq data provides a wealth of new information, allowing the identification of RPs of interest in many disease and normal contexts. dripARF is available as part of the ARF R package and can be accessed through https://github.com/fallerlab/ARF.
- Published
- 2022
5. An improved iCLIP protocol
- Author
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Heike Hänel, Christopher R. Sibley, Martina Hallegger, Martin R Turner, Patrick Toolan-Kerr, Christoph Sadee, Federica Capraro, Julian König, Cristina Militti, Flora C.Y. Lee, Elisa Monzón-Casanova, Anob M. Chakrabarti, Oscar G. Wilkins, and Jernej Ule
- Subjects
Exon ,Immunoprecipitation ,Computer science ,cDNA library ,RNA ,RNA-binding protein ,Computational biology ,PTBP1 ,Binding site ,ICLIP - Abstract
Crosslinking and Immunoprecipitation (CLIP) is a powerful technique to obtain transcriptome-wide maps of in vivo protein-RNA interactions, which are important to understand the post-transcriptional mechanisms mediated by RNA binding proteins (RBPs). Many variant CLIP protocols have been developed to improve the efficiency and convenience of cDNA library preparation. Here we describe an improved individual nucleotide resolution CLIP protocol (iiCLIP), which can be completed within 4 days from UV crosslinking to libraries for sequencing. For benchmarking, we directly compared PTBP1 iiCLIP libraries with the iCLIP2 protocol produced under standardised conditions, and with public eCLIP and iCLIP PTBP1 data. We visualised enriched motifs surrounding the identified crosslink positions and RNA maps of these crosslinks around the alternative exons regulated by PTBP1. Notably, motif enrichment was higher in iiCLIP and iCLIP2 in comparison to public eCLIP and iCLIP, and we show how this impacts the specificity of RNA maps. In conclusion, iiCLIP is technically convenient and efficient, and enables production of highly specific datasets for identifying RBP binding sites.
- Published
- 2021
6. FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation
- Author
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Oscar G. Wilkins, Pietro Fratta, Seth Jarvis, Brian Tsang, Elizabeth M. C. Fisher, Cristian Bodo, Giampietro Schiavo, Maria Giovanna Garone, Anny Devoy, Gabriella Viero, Julie D. Forman-Kay, Micheal L. Nosella, P. Andrew Chong, Melis Pisiren, Agnieszka M. Ule, Nicol Birsa, Francesca Mattedi, Alessandro Rosa, Jack Humphrey, and Rafaela Fernandez de la Fuente
- Subjects
0301 basic medicine ,congenital, hereditary, and neonatal diseases and abnormalities ,RNA-binding protein ,Mutant ,Phase separation ,Neurodegenerative diseases ,Amyotrophic lateral sclerosis ,Fragile X Mental Retardation Protein ,Mice ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,Brain ,Cell proliferation ,Neurons ,Proteins ,Psychological repression ,Research Articles ,Multidisciplinary ,Chemistry ,Neurodegeneration ,SciAdv r-articles ,Translation (biology) ,medicine.disease ,In vitro ,nervous system diseases ,Cell biology ,030104 developmental biology ,Cytoplasm ,Protein Biosynthesis ,Cellular Neuroscience ,Mutation ,RNA-Binding Protein FUS ,030217 neurology & neurosurgery ,Research Article - Abstract
Cytoplasmic mislocalization of FUS-ALS mutants determines aberrant FMRP condensates and protein synthesis repression., FUsed in Sarcoma (FUS) is a multifunctional RNA binding protein (RBP). FUS mutations lead to its cytoplasmic mislocalization and cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we use mouse and human models with endogenous ALS-associated mutations to study the early consequences of increased cytoplasmic FUS. We show that in axons, mutant FUS condensates sequester and promote the phase separation of fragile X mental retardation protein (FMRP), another RBP associated with neurodegeneration. This leads to repression of translation in mouse and human FUS-ALS motor neurons and is corroborated in vitro, where FUS and FMRP copartition and repress translation. Last, we show that translation of FMRP-bound RNAs is reduced in vivo in FUS-ALS motor neurons. Our results unravel new pathomechanisms of FUS-ALS and identify a novel paradigm by which mutations in one RBP favor the formation of condensates sequestering other RBPs, affecting crucial biological functions, such as protein translation.
- Published
- 2021
7. MIR-NATs repress MAPT translation and aid proteostasis in neurodegeneration
- Author
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Filipa Almeida, Kavitha Siva, Oscar G. Wilkins, Gurvir S. Virdi, Warren Emmett, Elisavet Preza, Michela A. Denti, Jernej Ule, Claudia Manzoni, Alessandro Quattrone, Daniah Trabzuni, Victoria Kay, Mina Ryten, Thomas T. Warner, Roberto Simone, Jamie S. Mitchell, Natalia Barahona-Torres, Demis A. Kia, Per Svenningsson, Mazdak Ehteramyan, Andrew J. Lees, Raffaele Ferrari, Angelika Modelska, Geshanthi Hondhamuni, Jasmine Harley, Rohan de Silva, Vincent Plagnol, Rickie Patani, Alan Pittman, Justyna Zareba-Paslawska, John Hardy, Faiza Javad, Paola Zuccotti, and Selina Wray
- Subjects
Male ,Mice, Transgenic ,tau Proteins ,Biology ,Internal Ribosome Entry Sites ,Transgenic ,Article ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Transcription (biology) ,medicine ,Gene silencing ,Animals ,Humans ,RNA, Antisense ,Antisense ,Gene ,030304 developmental biology ,Aged ,Neurons ,0303 health sciences ,Multidisciplinary ,Binding Sites ,Brain ,Case-Control Studies ,Cell Differentiation ,Disease Progression ,Female ,Middle Aged ,Protein Biosynthesis ,Proteostasis ,Ribosomes ,Tauopathies ,Neurodegeneration ,fungi ,RNA ,Translation (biology) ,medicine.disease ,Cell biology ,Human genome ,030217 neurology & neurosurgery - Abstract
The human genome expresses thousands of natural antisense transcripts (NAT) that can regulate epigenetic state, transcription, RNA stability or translation of their overlapping genes1,2. Here we describe MAPT-AS1, a brain-enriched NAT that is conserved in primates and contains an embedded mammalian-wide interspersed repeat (MIR), which represses tau translation by competing for ribosomal RNA pairing with the MAPT mRNA internal ribosome entry site3. MAPT encodes tau, a neuronal intrinsically disordered protein (IDP) that stabilizes axonal microtubules. Hyperphosphorylated, aggregation-prone tau forms the hallmark inclusions of tauopathies4. Mutations in MAPT cause familial frontotemporal dementia, and common variations forming the MAPT H1 haplotype are a significant risk factor in many tauopathies5 and Parkinson's disease. Notably, expression of MAPT-AS1 or minimal essential sequences from MAPT-AS1 (including MIR) reduces-whereas silencing MAPT-AS1 expression increases-neuronal tau levels, and correlate with tau pathology in human brain. Moreover, we identified many additional NATs with embedded MIRs (MIR-NATs), which are overrepresented at coding genes linked to neurodegeneration and/or encoding IDPs, and confirmed MIR-NAT-mediated translational control of one such gene, PLCG1. These results demonstrate a key role for MAPT-AS1 in tauopathies and reveal a potentially broad contribution of MIR-NATs to the tightly controlled translation of IDPs6, with particular relevance for proteostasis in neurodegeneration.
- Published
- 2021
8. Ultraplex: A rapid, flexible, all-in-one fastq demultiplexer
- Author
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Jernej Ule, Nicholas M. Luscombe, Oscar G. Wilkins, and Charlotte Capitanchik
- Subjects
FASTQ format ,Demultiplexer ,Computer science ,iCLIP ,Medicine (miscellaneous) ,General Biochemistry, Genetics and Molecular Biology ,DNA sequencing ,03 medical and health sciences ,0302 clinical medicine ,Software ,fastq ,Header ,Demultiplexing ,Ribosome profiling ,030304 developmental biology ,ribosome profiling ,0303 health sciences ,business.industry ,Software Tool Article ,Articles ,UMI ,Pipeline (software) ,Identifier ,030220 oncology & carcinogenesis ,business ,Computer hardware - Abstract
Background: The first step of virtually all next generation sequencing analysis involves the splitting of the raw sequencing data into separate files using sample-specific barcodes, a process known as “demultiplexing”. However, we found that existing software for this purpose was either too inflexible or too computationally intensive for fast, streamlined processing of raw, single end fastq files containing combinatorial barcodes. Results: Here, we introduce a fast and uniquely flexible demultiplexer, named Ultraplex, which splits a raw FASTQ file containing barcodes either at a single end or at both 5’ and 3’ ends of reads, trims the sequencing adaptors and low-quality bases, and moves unique molecular identifiers (UMIs) into the read header, allowing subsequent removal of PCR duplicates. Ultraplex is able to perform such single or combinatorial demultiplexing on both single- and paired-end sequencing data, and can process an entire Illumina HiSeq lane, consisting of nearly 500 million reads, in less than 20 minutes. Conclusions: Ultraplex greatly reduces computational burden and pipeline complexity for the demultiplexing of complex sequencing libraries, such as those produced by various CLIP and ribosome profiling protocols, and is also very user friendly, enabling streamlined, robust data processing. Ultraplex is available on PyPi and Conda and via Github.
- Published
- 2021
9. Common ALS/FTD risk variants in UNC13A exacerbate its cryptic splicing and loss upon TDP-43 mislocalization
- Author
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Tammaryn Lashley, Maria Secrier, Giampietro Schiavo, Pietro Fratta, Hemali Phatnani, Flora C.Y. Lee, Sam Byrce-Smith, Anna-Leigh Brown, Matthew Keuss, Yue Qi, Laura Masino, Sarah C. Hill, Ariana Gatt, Alexander Bampton, Towfique Raj, Michael E. Ward, Elizabeth M. C. Fisher, Jernej Ule, Emanuele Buratti, Jack Humphrey, Oscar G. Wilkins, Weaverly Colleen Lee, and Matteo Zanovello
- Subjects
Genetics ,Mechanism (biology) ,Intron ,nutritional and metabolic diseases ,Disease ,Biology ,medicine.disease ,nervous system diseases ,mental disorders ,medicine ,Cryptic splicing ,Amyotrophic lateral sclerosis ,Gene ,Function (biology) ,Frontotemporal dementia - Abstract
Variants within the UNC13A gene have long been known to increase risk of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-43. Here, we show that TDP-43 depletion induces robust inclusion of a cryptic exon (CE) within UNC13A, a critical synaptic gene, resulting in nonsense-mediated decay and protein loss. Strikingly, two common polymorphisms strongly associated with ALS/FTD risk directly alter TDP-43 binding within the CE or downstream intron, increasing CE inclusion in cultured cells and in patient brains. Our findings, which are the first to demonstrate a genetic link specifically between loss of TDP-43 nuclear function and disease, reveal both the mechanism by which UNC13A variants exacerbate the effects of decreased nuclear TDP-43 function, and provide a promising therapeutic target for TDP-43 proteinopathies. One-Sentence Summary Shared ALS/FTD risk variants increase the sensitivity of a cryptic exon in the synaptic gene UNC13A to TDP-43 depletion.
- Published
- 2021
10. FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation
- Author
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Michael L Nosella, M. Pisiren, R. Fernandez de la Fuente, Brian Tsang, Gabriella Viero, Cristian Bodo, Pietro Fratta, Maria Giovanna Garone, Giampietro Schiavo, Alessandro Rosa, Francesca Mattedi, Oscar G. Wilkins, Elizabeth M. C. Fisher, Agnieszka M. Ule, Jack Humphrey, Seth Jarvis, Julie D. Forman-Kay, P.A. Chong, Nicol Birsa, and Anny Devoy
- Subjects
Cytoplasm ,Chemistry ,Mutant ,Translational regulation ,Neurodegeneration ,medicine ,RNA ,RNA-binding protein ,Translation (biology) ,medicine.disease ,Ribosome ,Cell biology - Abstract
SummaryMutations in the RNA binding protein (RBP) FUS cause amyotrophic lateral sclerosis (ALS) and result in its nuclear depletion and cytoplasmic mislocalisation, with cytoplasmic gain of function thought to be crucial in pathogenesis. Here, we show that expression of mutant FUS at physiological levels drives translation inhibition in both mouse and human motor neurons. Rather than acting directly on the translation machinery, we find that mutant FUS forms cytoplasmic condensates that promote the phase separation of FMRP, another RBP associated with neurodegeneration and robustly involved in translation regulation. FUS and FMRP co-partition and repress translation in vitro. In our in vivo model, FMRP RNA targets are depleted from ribosomes. Our results identify a novel paradigm by which FUS mutations favour the condensed state of other RBPs, impacting on crucial biological functions, such as protein translation.
- Published
- 2020
11. Highly disordered histone H1-DNA model complexes and their condensates
- Author
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Oscar G. Wilkins, Jean O. Thomas, Katherine Stott, Andrew Travers, Abigail L. Turner, Matthew Watson, Laura Cato, Stott, Katherine [0000-0002-4014-1188], and Apollo - University of Cambridge Repository
- Subjects
0301 basic medicine ,Magnetic Resonance Spectroscopy ,Protein Conformation ,histone H1 ,Histones ,03 medical and health sciences ,chemistry.chemical_compound ,Prophase ,Histone H1 ,Animals ,Humans ,Multidisciplinary ,Coacervate ,030102 biochemistry & molecular biology ,Chemistry ,phosphorylation ,intrinsic disorder ,DNA ,Chromatin Assembly and Disassembly ,Linker DNA ,Chromatin ,DNA-Binding Proteins ,030104 developmental biology ,Biophysics ,Phosphorylation ,Nucleic Acid Conformation ,phase separation ,Protein Processing, Post-Translational ,Macromolecule ,Protein Binding - Abstract
Disordered proteins play an essential role in a wide variety of biological processes, and are often posttranslationally modified. One such protein is histone H1; its highly disordered C-terminal tail (CH1) condenses internucleosomal linker DNA in chromatin in a way that is still poorly understood. Moreover, CH1 is phosphorylated in a cell cycle-dependent manner that correlates with changes in the chromatin condensation level. Here we present a model system that recapitulates key aspects of the in vivo process, and also allows a detailed structural and biophysical analysis of the stages before and after condensation. CH1 remains disordered in the DNA-bound state, despite its nanomolar affinity. Phase-separated droplets (coacervates) form, containing higher-order assemblies of CH1/DNA complexes. Phosphorylation at three serine residues, spaced along the length of the tail, has little effect on the local properties of the condensate. However, it dramatically alters higher-order structure in the coacervate and reduces partitioning to the coacervate phase. These observations show that disordered proteins can bind tightly to DNA without a disorder-to-order transition. Importantly, they also provide mechanistic insights into how higher-order structures can be exquisitely sensitive to perturbation by posttranslational modifications, thus broadening the repertoire of mechanisms that might regulate chromatin and other macromolecular assemblies.
- Published
- 2018
- Full Text
- View/download PDF
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