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Sudhakaran Prabakaran - One of the best experts on this subject based on the ideXlab platform.
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in silico identification of novel Open Reading Frames in plasmodium falciparum oocyte and salivary gland sporozoites using proteogenomics framework
Malaria Journal, 2021Co-Authors: Sophie Gunnarsson, Sudhakaran PrabakaranAbstract:Background Plasmodium falciparum causes the deadliest form of malaria, which remains one of the most prevalent infectious diseases. Unfortunately, the only licensed vaccine showed limited protection and resistance to anti-malarial drug is increasing, which can be largely attributed to the biological complexity of the parasite's life cycle. The progression from one developmental stage to another in P. falciparum involves drastic changes in gene expressions, where its infectivity to human hosts varies greatly depending on the stage. Approaches to identify candidate genes that are responsible for the development of infectivity to human hosts typically involve differential gene expression analysis between stages. However, the detection may be limited to annotated proteins and Open Reading Frames (ORFs) predicted using restrictive criteria. Methods The above problem is particularly relevant for P. falciparum; whose genome annotation is relatively incomplete given its clinical significance. In this work, systems proteogenomics approach was used to address this challenge, as it allows computational detection of unannotated, novel Open Reading Frames (nORFs), which are neglected by conventional analyses. Two pairs of transcriptome/proteome were obtained from a previous study where one was collected in the mosquito-infectious oocyst sporozoite stage, and the other in the salivary gland sporozoite stage with human infectivity. They were then re-analysed using the proteogenomics framework to identify nORFs in each stage. Results Translational products of nORFs that map to antisense, intergenic, intronic, 3' UTR and 5' UTR regions, as well as alternative Reading Frames of canonical proteins were detected. Some of these nORFs also showed differential expression between the two life cycle stages studied. Their regulatory roles were explored through further bioinformatics analyses including the expression regulation on the parent reference genes, in silico structure prediction, and gene ontology term enrichment analysis. Conclusion The identification of nORFs in P. falciparum sporozoites highlights the biological complexity of the parasite. Although the analyses are solely computational, these results provide a starting point for further experimental validation of the existence and functional roles of these nORFs.
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in silico identification of novel Open Reading Frames in plasmodium falciparum oocyte and salivary gland sporozoites using proteogenomics framework
Malaria Journal, 2021Co-Authors: Sophie Gunnarsson, Sudhakaran PrabakaranAbstract:Plasmodium falciparum causes the deadliest form of malaria, which remains one of the most prevalent infectious diseases. Unfortunately, the only licensed vaccine showed limited protection and resistance to anti-malarial drug is increasing, which can be largely attributed to the biological complexity of the parasite’s life cycle. The progression from one developmental stage to another in P. falciparum involves drastic changes in gene expressions, where its infectivity to human hosts varies greatly depending on the stage. Approaches to identify candidate genes that are responsible for the development of infectivity to human hosts typically involve differential gene expression analysis between stages. However, the detection may be limited to annotated proteins and Open Reading Frames (ORFs) predicted using restrictive criteria. The above problem is particularly relevant for P. falciparum; whose genome annotation is relatively incomplete given its clinical significance. In this work, systems proteogenomics approach was used to address this challenge, as it allows computational detection of unannotated, novel Open Reading Frames (nORFs), which are neglected by conventional analyses. Two pairs of transcriptome/proteome were obtained from a previous study where one was collected in the mosquito-infectious oocyst sporozoite stage, and the other in the salivary gland sporozoite stage with human infectivity. They were then re-analysed using the proteogenomics framework to identify nORFs in each stage. Translational products of nORFs that map to antisense, intergenic, intronic, 3′ UTR and 5′ UTR regions, as well as alternative Reading Frames of canonical proteins were detected. Some of these nORFs also showed differential expression between the two life cycle stages studied. Their regulatory roles were explored through further bioinformatics analyses including the expression regulation on the parent reference genes, in silico structure prediction, and gene ontology term enrichment analysis. The identification of nORFs in P. falciparum sporozoites highlights the biological complexity of the parasite. Although the analyses are solely computational, these results provide a starting point for further experimental validation of the existence and functional roles of these nORFs,
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a platform for curated products from novel Open Reading Frames prompts reinterpretation of disease variants
Genome Research, 2021Co-Authors: Matthew D C Neville, Sudhakaran Prabakaran, Chaitanya Erady, Narendra Meena, Robin Kohze, Matthew J Hayden, David Neil Cooper, Matthew MortAbstract:Recent evidence from proteomics and deep massively parallel sequencing studies have revealed that eukaryotic genomes contain substantial numbers of as-yet-uncharacterized Open Reading Frames (ORFs). We define these uncharacterized ORFs as novel ORFs (nORFs). nORFs in humans are mostly under 100 codons and are found in diverse regions of the genome, including in long noncoding RNAs, pseudogenes, 3' UTRs, 5' UTRs, and alternative Reading Frames of canonical protein coding exons. There is therefore a pressing need to evaluate the potential functional importance of these unannotated transcripts and proteins in biological pathways and human disease on a larger scale, rather than one at a time. In this study, we outline the creation of a valuable nORFs data set with experimental evidence of translation for the community, use measures of heritability and selection that reveal signals for functional importance, and show the potential implications for functional interpretation of genetic variants in nORFs. Our results indicate that some variants that were previously classified as being benign or of uncertain significance may have to be reinterpreted.
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Evolutionary divergence of novel Open Reading Frames in cichlids speciation
2020Co-Authors: Shraddha Puntambekar, Rachel Newhouse, Jaime San Miguel Navas, Ruchi Chauhan, Gregoire Vernaz, Thomas Willis, Matthew T. Wayland, Yagnesh Urmania, Eric A. Miska, Sudhakaran PrabakaranAbstract:Novel Open Reading Frames (nORFs) with coding potential may arise from noncoding DNA. Not much is known about their emergence, functional role, fixation in a population or contribution to adaptive radiation. Cichlids fishes exhibit extensive phenotypic diversification and speciation. Encounters with new environments alone are not sufficient to explain this striking diversity of cichlid radiation because other taxa coexistent with the Cichlidae demonstrate lower species richness. Wagner et al analyzed cichlid diversification in 46 African lakes and reported that both extrinsic environmental factors and intrinsic lineage-specific traits related to sexual selection 1 have strongly influenced the cichlid radiation ,which indicates the existence of unknown molecular mechanisms responsible for rapid phenotypic diversification, such as emergence of novel Open Reading Frames (nORFs). In this study, we integrated transcriptomic and proteomic signatures from two tissues of two cichlids species, identified nORFs and performed evolutionary analysis on these nORF regions. Our results suggest that the time scale of speciation of the two species and evolutionary divergence of these nORF genomic regions are similar and indicate a potential role for these nORFs in speciation of the cichlid fishes.
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rapid speciation of cichlids fishes may be explained by evolutionary divergence of novel Open Reading Frames
bioRxiv, 2020Co-Authors: Shraddha Puntambekar, Rachel Newhouse, Ruchi Chauhan, Gregoire Vernaz, Thomas Willis, Matthew T. Wayland, Yagnesh Urmania, Eric A. Miska, Jaime San Miguel Navas, Sudhakaran PrabakaranAbstract:Cichlids fishes exhibit extensive phenotypic diversification and speciation. Encounters with new environments alone are not sufficient to explain this striking diversity of cichlid radiation because other taxa coexistent with the Cichlidae demonstrate lower species richness. Wagner et al analyzed cichlid diversification in 46 African lakes and reported that both extrinsic environmental factors and intrinsic lineage-specific traits related to sexual selection have strongly influenced the cichlid radiation 1 , which indicates the existence of molecular mechanisms responsible for rapid phenotypic diversification and events leading to reproductive isolation. However discovery of the molecular mechanisms in terms of genetic or transcriptomic or proteomic diversity responsible for the rapid speciation in a short geological time has remained elusive. In this study we integrated transcriptomic and proteomic signatures from two cichlids species, identified novel Open Reading Frames (nORFs) and performed evolutionary analysis on these nORF regions. Our results suggest that the time scale of speciation of the two species can be better explained by the evolutionary divergence of these nORF genomic regions. Therefore this study has revealed the potential functional and evolutionary role of nORFs, which has far reaching implications for such evolutionary and speciation studies that have traditionally been focussed on known protein coding regions.
Juan Pablo Couso - One of the best experts on this subject based on the ideXlab platform.
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classification and function of small Open Reading Frames
Nature Reviews Molecular Cell Biology, 2017Co-Authors: Juan Pablo Couso, Pedro PatraquimAbstract:Small Open Reading Frames (smORFs) of 100 codons or fewer are usually - if arbitrarily - excluded from proteome annotations. Despite this, the genomes of many metazoans, including humans, contain millions of smORFs, some of which fulfil key physiological functions. Recently, the transcriptome of Drosophila melanogaster was shown to contain thousands of smORFs of different classes that actively undergo translation, which produces peptides of mostly unknown function. Here, we present a comprehensive analysis of smORFs in flies, mice and humans. We propose the existence of several functional classes of smORFs, ranging from inert DNA sequences to transcribed and translated cis-regulators of translation and peptides with a prOpensity to function as regulators of membrane-associated proteins, or as components of ancient protein complexes in the cytoplasm. We suggest that the different smORF classes could represent steps in gene, peptide and protein evolution. Our analysis introduces a distinction between different peptide-coding classes of smORFs in animal genomes, and highlights the role of model organisms for the study of small peptide biology in the context of development, physiology and human disease.
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extensive translation of small Open Reading Frames revealed by poly ribo seq
eLife, 2014Co-Authors: Julie L Aspden, Ying Chen Eyrewalker, Rose Phillips, Unum Amin, Muhammad Ali Shahzad Mumtaz, Michele Brocard, Juan Pablo CousoAbstract:Thousands of small Open Reading Frames (smORFs) with the potential to encode small peptides of fewer than 100 amino acids exist in our genomes. However, the number of smORFs actually translated, and their molecular and functional roles are still unclear. In this study, we present a genome-wide assessment of smORF translation by ribosomal profiling of polysomal fractions in Drosophila. We detect two types of smORFs bound by multiple ribosomes and thus undergoing productive translation. The 'longer' smORFs of around 80 amino acids resemble canonical proteins in translational metrics and conservation, and display a prOpensity to contain transmembrane motifs. The 'dwarf' smORFs are in general shorter (around 20 amino-acid long), are mostly found in 5'-UTRs and non-coding RNAs, are less well conserved, and have no bioinformatic indicators of peptide function. Our findings indicate that thousands of smORFs are translated in metazoan genomes, reinforcing the idea that smORFs are an abundant and fundamental genome component.
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conserved regulation of cardiac calcium uptake by peptides encoded in small Open Reading Frames
Science, 2013Co-Authors: Emile G Magny, Jose Ignacio Pueyo, Frances M G Pearl, Miguel Angel Cespedes, Jeremy E Niven, Sarah A Bishop, Juan Pablo CousoAbstract:Small Open Reading Frames (smORFs) are short DNA sequences that are able to encode small peptides of less than 100 amino acids. Study of these elements has been neglected despite thousands existing in our genomes. We and others previously showed that peptides as short as 11 amino acids are translated and provide essential functions during insect development. Here, we describe two peptides of less than 30 amino acids regulating calcium transport, and hence influencing regular muscle contraction, in the Drosophila heart. These peptides seem conserved for more than 550 million years in a range of species from flies to humans, in which they have been implicated in cardiac pathologies. Such conservation suggests that the mechanisms for heart regulation are ancient and that smORFs may be a fundamental genome component that should be studied systematically.
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hundreds of putatively functional small Open Reading Frames in drosophila
Genome Biology, 2011Co-Authors: Emmanuel D Ladoukakis, Vini Pereira, Emile G Magny, Adam Eyrewalker, Juan Pablo CousoAbstract:Background The relationship between DNA sequence and encoded information is still an unsolved puzzle. The number of protein-coding genes in higher eukaryotes identified by genome projects is lower than was expected, while a considerable amount of putatively non-coding transcription has been detected. Functional small Open Reading Frames (smORFs) are known to exist in several organisms. However, coding sequence detection methods are biased against detecting such very short Open Reading Frames. Thus, a substantial number of non-canonical coding regions encoding short peptides might await characterization.
Sophie Gunnarsson - One of the best experts on this subject based on the ideXlab platform.
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in silico identification of novel Open Reading Frames in plasmodium falciparum oocyte and salivary gland sporozoites using proteogenomics framework
Malaria Journal, 2021Co-Authors: Sophie Gunnarsson, Sudhakaran PrabakaranAbstract:Background Plasmodium falciparum causes the deadliest form of malaria, which remains one of the most prevalent infectious diseases. Unfortunately, the only licensed vaccine showed limited protection and resistance to anti-malarial drug is increasing, which can be largely attributed to the biological complexity of the parasite's life cycle. The progression from one developmental stage to another in P. falciparum involves drastic changes in gene expressions, where its infectivity to human hosts varies greatly depending on the stage. Approaches to identify candidate genes that are responsible for the development of infectivity to human hosts typically involve differential gene expression analysis between stages. However, the detection may be limited to annotated proteins and Open Reading Frames (ORFs) predicted using restrictive criteria. Methods The above problem is particularly relevant for P. falciparum; whose genome annotation is relatively incomplete given its clinical significance. In this work, systems proteogenomics approach was used to address this challenge, as it allows computational detection of unannotated, novel Open Reading Frames (nORFs), which are neglected by conventional analyses. Two pairs of transcriptome/proteome were obtained from a previous study where one was collected in the mosquito-infectious oocyst sporozoite stage, and the other in the salivary gland sporozoite stage with human infectivity. They were then re-analysed using the proteogenomics framework to identify nORFs in each stage. Results Translational products of nORFs that map to antisense, intergenic, intronic, 3' UTR and 5' UTR regions, as well as alternative Reading Frames of canonical proteins were detected. Some of these nORFs also showed differential expression between the two life cycle stages studied. Their regulatory roles were explored through further bioinformatics analyses including the expression regulation on the parent reference genes, in silico structure prediction, and gene ontology term enrichment analysis. Conclusion The identification of nORFs in P. falciparum sporozoites highlights the biological complexity of the parasite. Although the analyses are solely computational, these results provide a starting point for further experimental validation of the existence and functional roles of these nORFs.
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in silico identification of novel Open Reading Frames in plasmodium falciparum oocyte and salivary gland sporozoites using proteogenomics framework
Malaria Journal, 2021Co-Authors: Sophie Gunnarsson, Sudhakaran PrabakaranAbstract:Plasmodium falciparum causes the deadliest form of malaria, which remains one of the most prevalent infectious diseases. Unfortunately, the only licensed vaccine showed limited protection and resistance to anti-malarial drug is increasing, which can be largely attributed to the biological complexity of the parasite’s life cycle. The progression from one developmental stage to another in P. falciparum involves drastic changes in gene expressions, where its infectivity to human hosts varies greatly depending on the stage. Approaches to identify candidate genes that are responsible for the development of infectivity to human hosts typically involve differential gene expression analysis between stages. However, the detection may be limited to annotated proteins and Open Reading Frames (ORFs) predicted using restrictive criteria. The above problem is particularly relevant for P. falciparum; whose genome annotation is relatively incomplete given its clinical significance. In this work, systems proteogenomics approach was used to address this challenge, as it allows computational detection of unannotated, novel Open Reading Frames (nORFs), which are neglected by conventional analyses. Two pairs of transcriptome/proteome were obtained from a previous study where one was collected in the mosquito-infectious oocyst sporozoite stage, and the other in the salivary gland sporozoite stage with human infectivity. They were then re-analysed using the proteogenomics framework to identify nORFs in each stage. Translational products of nORFs that map to antisense, intergenic, intronic, 3′ UTR and 5′ UTR regions, as well as alternative Reading Frames of canonical proteins were detected. Some of these nORFs also showed differential expression between the two life cycle stages studied. Their regulatory roles were explored through further bioinformatics analyses including the expression regulation on the parent reference genes, in silico structure prediction, and gene ontology term enrichment analysis. The identification of nORFs in P. falciparum sporozoites highlights the biological complexity of the parasite. Although the analyses are solely computational, these results provide a starting point for further experimental validation of the existence and functional roles of these nORFs,
Joseph A Rothnagel - One of the best experts on this subject based on the ideXlab platform.
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emerging evidence for functional peptides encoded by short Open Reading Frames
Nature Reviews Genetics, 2014Co-Authors: Shea J Andrews, Joseph A RothnagelAbstract:Short Open Reading Frames (sORFs) are a common feature of all genomes, but their coding potential has mostly been disregarded, partly because of the difficulty in determining whether these sequences are translated. Recent innovations in computing, proteomics and high-throughput analyses of translation start sites have begun to address this challenge and have identified hundreds of putative coding sORFs. The translation of some of these has been confirmed, although the contribution of their peptide products to cellular functions remains largely unknown. This Review examines this hitherto overlooked component of the proteome and considers potential roles for sORF-encoded peptides.
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evidence for conservation and selection of upstream Open Reading Frames suggests probable encoding of bioactive peptides
BMC Genomics, 2006Co-Authors: Mark L Crowe, Xueqing Wang, Joseph A RothnagelAbstract:Approximately 40% of mammalian mRNA sequences contain AUG trinucleotides upstream of the main coding sequence, with a quarter of these AUGs demarcating Open Reading Frames of 20 or more codons. In order to investigate whether these Open Reading Frames may encode functional peptides, we have carried out a comparative genomic analysis of human and mouse mRNA 'untranslated regions' using sequences from the RefSeq mRNA sequence database. We have identified over 200 upstream Open Reading Frames which are strongly conserved between the human and mouse genomes. Consensus sequences associated with efficient initiation of translation are overrepresented at the AUG trinucleotides of these upstream Open Reading Frames, while comparative analysis of their DNA and putative peptide sequences shows evidence of purifying selection. The occurrence of a large number of conserved upstream Open Reading Frames, in association with features consistent with protein translation, strongly suggests evolutionary maintenance of the coding sequence and indicates probable functional expression of the peptides encoded within these upstream Open Reading Frames.
John R. Prensner - One of the best experts on this subject based on the ideXlab platform.
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a community driven roadmap to advance research on translated Open Reading Frames detected by ribo seq
bioRxiv, 2021Co-Authors: Jonathan M Mudge, John R. Prensner, Thomas F Martinez, Jorge Ruizorera, Jose Manuel Gonzalez, Michele Magrane, Jana Felicitas Schulz, Yi YangAbstract:Ribosome profiling (Ribo-seq) has catalyzed a paradigm shift in our understanding of the translational ‘vocabulary’ of the human genome, discovering thousands of translated Open Reading Frames (ORFs) within long non-coding RNAs and presumed untranslated regions of protein-coding genes. However, reference gene annotation projects have been circumspect in their incorporation of these ORFs due to uncertainties about their experimental reproducibility and physiological roles. Yet, it is indisputable that certain Ribo-seq ORFs make stable proteins, others mediate gene regulation, and many have medical implications. Ultimately, the absence of standardized ORF annotation has created a circular problem: while Ribo-seq ORFs remain unannotated by reference biological databases, this lack of characterisation will thwart research efforts examining their roles. Here, we outline the initial stages of a community-led effort supported by GENCODE / Ensembl, HGNC and UniProt to produce a consolidated catalog of human Ribo-seq ORFs.
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Noncanonical Open Reading Frames encode functional proteins essential for cancer cell survival
Nature Biotechnology, 2021Co-Authors: John R. Prensner, Oana M. Enache, Victor Luria, Karsten Krug, Karl R. Clauser, Joshua M. Dempster, Amir Karger, Li Wang, Karolina Stumbraite, Vickie M. WangAbstract:Noncanonical Open Reading Frames are shown to be essential for cancer cell function. Although genomic analyses predict many noncanonical Open Reading Frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here we experimentally interrogated 553 candidates selected from noncanonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Following ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. Clustered regularly interspaced short palindromic repeat (CRISPR) tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442 —renamed g lycine-rich extracellular protein-1 (GREP1)—encodes a secreted protein highly expressed in breast cancer, and its knockout in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. Our experiments suggest that noncanonical ORFs can express biologically active proteins that are potential therapeutic targets.
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non canonical Open Reading Frames encode functional proteins essential for cancer cell survival
bioRxiv, 2020Co-Authors: John R. Prensner, Oana M. Enache, Victor Luria, Karsten Krug, Karl R. Clauser, Joshua M. Dempster, Amir Karger, Li WangAbstract:A key question in genome research is whether biologically active proteins are restricted to the ∼20,000 canonical, well-annotated genes, or rather extend to the many non-canonical Open Reading Frames (ORFs) predicted by genomic analyses. To address this, we experimentally interrogated 553 ORFs nominated in ribosome profiling datasets. Of these 553 ORFs, 57 (10%) induced a viability defect when the endogenous ORF was knocked out using CRISPR/Cas9 in 8 human cancer cell lines, 257 (46%) showed evidence of protein translation when ectopically expressed in HEK293T cells, and 401 (73%) induced gene expression changes measured by transcriptional profiling following ectopic expression across 4 cell types. CRISPR tiling and start codon mutagenesis indicated that the biological effects of these non-canonical ORFs required their translation as opposed to RNA-mediated effects. We selected one of these ORFs, G029442--renamed GREP1 (Glycine-Rich Extracellular Protein-1)--for further characterization. We found that GREP1 encodes a secreted protein highly expressed in breast cancer, and its knock-out in 263 cancer cell lines showed preferential essentiality in breast cancer derived lines. Analysis of the secretome of GREP1-expressing cells showed increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth inhibitory effect of GREP1 knock-out. Taken together, these experiments suggest that the non-canonical ORFeome is surprisingly rich in biologically active proteins and potential cancer therapeutic targets deserving of further study.
