scholarly journals Transcriptional and Translational Landscape of Equine Torovirus

2018 ◽  
Vol 92 (17) ◽  
Author(s):  
Hazel Stewart ◽  
Katherine Brown ◽  
Adam M. Dinan ◽  
Nerea Irigoyen ◽  
Eric J. Snijder ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Rna Synthesis ◽  
Gastrointestinal Disease ◽  
Ribosome Profiling ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Comparative Genomic ◽  
Human Pathogens ◽  
Content Type ◽  
Reading Frames

ABSTRACT The genus Torovirus (subfamily Torovirinae, family Coronaviridae, order Nidovirales) encompasses a range of species that infect domestic ungulates, including cattle, sheep, goats, pigs, and horses, causing an acute self-limiting gastroenteritis. Using the prototype species equine torovirus (EToV), we performed parallel RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) to analyze the relative expression levels of the known torovirus proteins and transcripts, chimeric sequences produced via discontinuous RNA synthesis (a characteristic of the nidovirus replication cycle), and changes in host transcription and translation as a result of EToV infection. RNA sequencing confirmed that EToV utilizes a unique combination of discontinuous and nondiscontinuous RNA synthesis to produce its subgenomic RNAs (sgRNAs); indeed, we identified transcripts arising from both mechanisms that would result in sgRNAs encoding the nucleocapsid. Our ribosome profiling analysis revealed that ribosomes efficiently translate two novel CUG-initiated open reading frames (ORFs), located within the so-called 5′ untranslated region. We have termed the resulting proteins U1 and U2. Comparative genomic analysis confirmed that these ORFs are conserved across all available torovirus sequences, and the inferred amino acid sequences are subject to purifying selection, indicating that U1 and U2 are functionally relevant. This study provides the first high-resolution analysis of transcription and translation in this neglected group of livestock pathogens. IMPORTANCE Toroviruses infect cattle, goats, pigs, and horses worldwide and can cause gastrointestinal disease. There is no treatment or vaccine, and their ability to spill over into humans has not been assessed. These viruses are related to important human pathogens, including severe acute respiratory syndrome (SARS) coronavirus, and they share some common features; however, the mechanism that they use to produce sgRNA molecules differs. Here, we performed deep sequencing to determine how equine torovirus produces sgRNAs. In doing so, we also identified two previously unknown open reading frames “hidden” within the genome. Together these results highlight the similarities and differences between this domestic animal virus and related pathogens of humans and livestock.

scholarly journals The transcriptional and translational landscape of equine torovirus

2018 ◽  
Author(s):  
Hazel Stewart ◽  
Katherine Brown ◽  
Adam M. Dinan ◽  
Nerea Irigoyen ◽  
Eric J. Snijder ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Rna Synthesis ◽  
Gastrointestinal Disease ◽  
Genomic Analysis ◽  
Ribosome Profiling ◽  
Amino Acid Sequences ◽  
Comparative Genomic ◽  
Human Pathogens ◽  
Animal Virus ◽  
Subgenomic Rnas

AbstractThe genusTorovirus(subfamilyTorovirinae, familyCoronaviridae, orderNidovirales) encompasses a range of species that infect domestic ungulates including cattle, sheep, goats, pigs and horses, causing an acute self-limiting gastroenteritis. Using the prototype species equine torovirus (EToV) we performed parallel RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) to analyse the relative expression levels of the known torovirus proteins and transcripts, chimaeric sequences produced via discontinuous RNA synthesis (a characteristic of the nidovirus replication cycle) and changes in host transcription and translation as a result of EToV infection. RNA sequencing confirmed that EToV utilises a unique combination of discontinuous and non-discontinuous RNA synthesis to produce its subgenomic RNAs; indeed, we identified transcripts arising from both mechanisms that would result in sgRNAs encoding the nucleocapsid. Our ribosome profiling analysis revealed that ribosomes efficiently translate two novel CUG-initiated ORFs, located within the so-called 5’ UTR. We have termed the resulting proteins U1 and U2. Comparative genomic analysis confirmed that these ORFs are conserved across all available torovirus sequences and the inferred amino acid sequences are subject to purifying selection, indicating that U1 and U2 are functionally relevant. This study provides the first high-resolution analysis of transcription and translation in this neglected group of livestock pathogens.ImportanceToroviruses infect cattle, goats, pigs and horses worldwide and can cause gastrointestinal disease. There is no treatment or vaccine and their ability to spill over into humans has not been assessed. These viruses are related to important human pathogens including severe acute respiratory syndrome (SARS) coronavirus and they share some common features, however the mechanism that they use to produce subgenomic RNA molecules differs. Here we performed deep sequencing to determine how equine torovirus produces subgenomic RNAs. In doing so, we also identified two previously unknown open reading frames “hidden” within the genome. Together these results highlight the similarities and differences between this domestic animal virus and related pathogens of humans and livestock.

scholarly journals Identifying Small Proteins by Ribosome Profiling with Stalled Initiation Complexes

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Jeremy Weaver ◽  
Fuad Mohammad ◽  
Allen R. Buskirk ◽  
Gisela Storz
Keyword(s):  
Amino Acids ◽  
Model Organism ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Genomic Context ◽  
Content Type ◽  
New Genes ◽  
Small Proteins ◽  
Intergenic Regions ◽  
Reading Frames

ABSTRACTSmall proteins consisting of 50 or fewer amino acids have been identified as regulators of larger proteins in bacteria and eukaryotes. Despite the importance of these molecules, the total number of small proteins remains unknown because conventional annotation pipelines usually exclude small open reading frames (smORFs). We previously identified several dozen small proteins in the model organismEscherichia coliusing theoretical bioinformatic approaches based on sequence conservation and matches to canonical ribosome binding sites. Here, we present an empirical approach for discovering new proteins, taking advantage of recent advances in ribosome profiling in which antibiotics are used to trap newly initiated 70S ribosomes at start codons. This approach led to the identification of many novel initiation sites in intergenic regions inE. coli. We tagged 41 smORFs on the chromosome and detected protein synthesis for all but three. Not only are the corresponding genes intergenic but they are also found antisense to other genes, in operons, and overlapping other open reading frames (ORFs), some impacting the translation of larger downstream genes. These results demonstrate the utility of this method for identifying new genes, regardless of their genomic context.IMPORTANCEProteins comprised of 50 or fewer amino acids have been shown to interact with and modulate the functions of larger proteins in a range of organisms. Despite the possible importance of small proteins, the true prevalence and capabilities of these regulators remain unknown as the small size of the proteins places serious limitations on their identification, purification, and characterization. Here, we present a ribosome profiling approach with stalled initiation complexes that led to the identification of 38 new small proteins.

scholarly journals Comparative Genomic Analysis of Acinetobacter oleivorans DR1 To Determine Strain-Specific Genomic Regions and Gentisate Biodegradation

2011 ◽  
Vol 77 (20) ◽  
pp. 7418-7424 ◽  
Author(s):  
Jaejoon Jung ◽  
Eugene L. Madsen ◽  
Che Ok Jeon ◽  
Woojun Park
Keyword(s):  
Metabolic Pathways ◽  
Genomic Analysis ◽  
Metal Resistance ◽  
Genomic Region ◽  
Open Reading Frames ◽  
Comparative Genomic ◽  
Content Type ◽  
Large Size ◽  
Genomic Regions ◽  
Reading Frames

ABSTRACTThe comparative genomics ofAcinetobacter oleivoransDR1 assayed withA. baylyiADP1,A. calcoaceticusPHEA-2, andA. baumanniiATCC 17978 revealed that the incorporation of phage-related genomic regions and the absence of transposable elements have contributed to the large size (4.15 Mb) of the DR1 genome. A horizontally transferred genomic region and a higher proportion of transcriptional regulator- and signal peptide-coding genes were identified as characteristics of the DR1 genome. Incomplete glucose metabolism, metabolic pathways of aromatic compounds, biofilm formation, antibiotics and metal resistance, and natural competence genes were conserved in four compared genomes. Interestingly, only strain DR1 possesses gentisate 1,2-dioxygenase (nagI) and grows on gentisate, whereas other species cannot. Expression of thenagIgene was upregulated during gentisate utilization, and four downstream open reading frames (ORFs) were cotranscribed, supporting the notion that gentisate metabolism is a unique characteristic of strain DR1. The genomic analysis of strain DR1 provides additional insights into the function, ecology, and evolution ofAcinetobacterspecies.

scholarly journals OpenProt 2021: deeper functional annotation of the coding potential of eukaryotic genomes

2020 ◽  
Vol 49 (D1) ◽  
pp. D380-D388 ◽  
Author(s):  
Marie A Brunet ◽  
Jean-François Lucier ◽  
Maxime Levesque ◽  
Sébastien Leblanc ◽  
Jean-Francois Jacques ◽  
...  
Keyword(s):  
Confidence Score ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Supporting Evidence ◽  
Initial Release ◽  
Ncbi Refseq ◽  
Computational Resources ◽  
Analysis Platform ◽  
Eukaryotic Genomes ◽  
Reading Frames

Abstract OpenProt (www.openprot.org) is the first proteogenomic resource supporting a polycistronic annotation model for eukaryotic genomes. It provides a deeper annotation of open reading frames (ORFs) while mining experimental data for supporting evidence using cutting-edge algorithms. This update presents the major improvements since the initial release of OpenProt. All species support recent NCBI RefSeq and Ensembl annotations, with changes in annotations being reported in OpenProt. Using the 131 ribosome profiling datasets re-analysed by OpenProt to date, non-AUG initiation starts are reported alongside a confidence score of the initiating codon. From the 177 mass spectrometry datasets re-analysed by OpenProt to date, the unicity of the detected peptides is controlled at each implementation. Furthermore, to guide the users, detectability statistics and protein relationships (isoforms) are now reported for each protein. Finally, to foster access to deeper ORF annotation independently of one’s bioinformatics skills or computational resources, OpenProt now offers a data analysis platform. Users can submit their dataset for analysis and receive the results from the analysis by OpenProt. All data on OpenProt are freely available and downloadable for each species, the release-based format ensuring a continuous access to the data. Thus, OpenProt enables a more comprehensive annotation of eukaryotic genomes and fosters functional proteomic discoveries.

scholarly journals Translation of ABCE1 Is Tightly Regulated by Upstream Open Reading Frames in Human Colorectal Cells

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 911
Author(s):  
Joana Silva ◽  
Pedro Nina ◽  
Luísa Romão
Keyword(s):  
Translational Regulation ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Leader Sequence ◽  
Open Reading Frames ◽  
Regulatory Function ◽  
Coding Sequence ◽  
Abc Protein ◽  
Upstream Open Reading Frames ◽  
Reading Frames

ATP-binding cassette subfamily E member 1 (ABCE1) belongs to the ABC protein family of transporters; however, it does not behave as a drug transporter. Instead, ABCE1 actively participates in different stages of translation and is also associated with oncogenic functions. Ribosome profiling analysis in colorectal cancer cells has revealed a high ribosome occupancy in the human ABCE1 mRNA 5′-leader sequence, indicating the presence of translatable upstream open reading frames (uORFs). These cis-acting translational regulatory elements usually act as repressors of translation of the main coding sequence. In the present study, we dissect the regulatory function of the five AUG and five non-AUG uORFs identified in the human ABCE1 mRNA 5′-leader sequence. We show that the expression of the main coding sequence is tightly regulated by the ABCE1 AUG uORFs in colorectal cells. Our results are consistent with a model wherein uORF1 is efficiently translated, behaving as a barrier to downstream uORF translation. The few ribosomes that can bypass uORF1 (and/or uORF2) must probably initiate at the inhibitory uORF3 or uORF5 that efficiently repress translation of the main ORF. This inhibitory property is slightly overcome in conditions of endoplasmic reticulum stress. In addition, we observed that these potent translation-inhibitory AUG uORFs function equally in cancer and in non-tumorigenic colorectal cells, which is consistent with a lack of oncogenic function. In conclusion, we establish human ABCE1 as an additional example of uORF-mediated translational regulation and that this tight regulation contributes to control ABCE1 protein levels in different cell environments.

scholarly journals Disrupting upstream translation in mRNAs is associated with human disease

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. M. Lee ◽  
Joseph Park ◽  
Andrew Kromer ◽  
Aris Baras ◽  
Daniel J. Rader ◽  
...  
Keyword(s):  
Protein Expression ◽  
Biological Significance ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Protein Coding ◽  
Stop Codons ◽  
Human Genes ◽  
Strong Negative Selection ◽  
Disease Associations ◽  
Reading Frames

AbstractRibosome-profiling has uncovered pervasive translation in non-canonical open reading frames, however the biological significance of this phenomenon remains unclear. Using genetic variation from 71,702 human genomes, we assess patterns of selection in translated upstream open reading frames (uORFs) in 5’UTRs. We show that uORF variants introducing new stop codons, or strengthening existing stop codons, are under strong negative selection comparable to protein-coding missense variants. Using these variants, we map and validate gene-disease associations in two independent biobanks containing exome sequencing from 10,900 and 32,268 individuals, respectively, and elucidate their impact on protein expression in human cells. Our results suggest translation disrupting mechanisms relating uORF variation to reduced protein expression, and demonstrate that translation at uORFs is genetically constrained in 50% of human genes.

scholarly journals Characterization and Genome Analysis of Staphylococcus aureus Podovirus CSA13 and Its Anti-Biofilm Capacity

Viruses ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 54 ◽  
Author(s):  
Yoyeon Cha ◽  
Jihwan Chun ◽  
Bokyung Son ◽  
Sangryeol Ryu
Keyword(s):  
Staphylococcus Aureus ◽  
Biocontrol Agent ◽  
Genomic Analysis ◽  
Open Reading Frames ◽  
Experimental Setting ◽  
Human Pathogens ◽  
Bacteriophage Therapy ◽  
Chromosomal Structure ◽  
Inhibition Effect ◽  
Reading Frames

Staphylococcus aureus is one of the notable human pathogens that can be easily encountered in both dietary and clinical surroundings. Among various countermeasures, bacteriophage therapy is recognized as an alternative method for resolving the issue of antibiotic resistance. In the current study, bacteriophage CSA13 was isolated from a chicken, and subsequently, its morphology, physiology, and genomics were characterized. This Podoviridae phage displayed an extended host inhibition effect of up to 23 hours of persistence. Its broad host spectrum included methicillin susceptible S. aureus (MSSA), methicillin resistant S. aureus (MRSA), local S. aureus isolates, as well as non-aureus staphylococci strains. Moreover, phage CSA13 could successfully remove over 78% and 93% of MSSA and MRSA biofilms in an experimental setting, respectively. Genomic analysis revealed a 17,034 bp chromosome containing 18 predicted open reading frames (ORFs) without tRNAs, representing a typical chromosomal structure of the staphylococcal Podoviridae family. The results presented here suggest that phage CSA13 can be applicable as an effective biocontrol agent against S. aureus.

scholarly journals Adding toYersinia enterocoliticaGene Pool Diversity: Two Cryptic Plasmids from a Biotype 1A Isolate

2009 ◽  
Vol 2009 ◽  
pp. 1-10 ◽  
Author(s):  
Daniela Lepka ◽  
Tobias Kerrinnes ◽  
Evelyn Skiebe ◽  
Birgitt Hahn ◽  
Angelika Fruth ◽  
...  
Keyword(s):  
Hypothetical Protein ◽  
Replication Initiation ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Eukaryotic Cells ◽  
Base Pairs ◽  
Significant Similarity ◽  
Replication Initiation Protein ◽  
Cryptic Plasmids ◽  
Reading Frames

We report the nucleotide sequence of two novel cryptic plasmids (4357 and 14 662 base pairs) carried by aYersinia enterocoliticabiotype 1A strain isolated from pork. As distinguished from most biotype 1A strains, this isolate, designated 07-04449, exhibited adherence to eukaryotic cells. The smaller plasmid pYe4449-1 carries five attributable open reading frames (ORFs) encoding the first CcdA/CcdB-like antitoxin/toxin system described for aYersiniaplasmid, a RepA-like replication initiation protein, and mobilizing factors MobA and MobC. The deduced amino acid sequences showed highest similarity to proteins described inSalmonella(CcdA/B),Klebsiella(RepA), andPlesiomonas(MobA/C) indicating genomic fluidity among members of theEnterobacteriaceae. One additional ORF with unknown function, termed ORF5, was identified with an ancestry distinct from the rest of the plasmid. While the C+G content of ORF5 is 38.3%, the rest of pYe4449-1 shows a C+G content of 55.7%. The C+G content of the larger plasmid pYe4449-2 (54.9%) was similar to that of pYe4449-1 (53.7%) and differed from that of theY. enterocoliticagenome (47.3%). Of the 14 ORFs identified on pYe4449-2, only six ORFs showed significant similarity to database entries. For three of these ORFs likely functions could be ascribed: a TnpR-like resolvase and a phage replication protein, localized each on a low C+G island, and DNA primase TraC. Two ORFs of pYe4449-2, ORF3 and ORF7, seem to encode secretable proteins. Epitope-tagging of ORF3 revealed protein expression at4°Cbut not at or above27°Csuggesting adaptation to a habitat outside swine. The hypothetical protein encoded by ORF7 is the member of a novel repeat protein family sharing theDxxGN(x)nDxxGNmotif. Our findings illustrate the exceptional gene pool diversity within the speciesY. enterocoliticadriven by horizontal gene transfer events.

scholarly journals Two Unrelated 8-Vinyl Reductases Ensure Production of Mature Chlorophylls in Acaryochloris marina

2016 ◽  
Vol 198 (9) ◽  
pp. 1393-1400 ◽  
Author(s):  
Guangyu E. Chen ◽  
Andrew Hitchcock ◽  
Philip J. Jackson ◽  
Roy R. Chaudhuri ◽  
Mark J. Dickman ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Sequence Similarity ◽  
Open Reading Frames ◽  
High Sequence Similarity ◽  
Content Type ◽  
Ethyl Group ◽  
Acaryochloris Marina ◽  
Vinyl Group ◽  
Vinyl Reductase ◽  
Reading Frames

ABSTRACTThe major photopigment of the cyanobacteriumAcaryochloris marinais chlorophylld, while its direct biosynthetic precursor, chlorophylla, is also present in the cell. These pigments, along with the majority of chlorophylls utilized by oxygenic phototrophs, carry an ethyl group at the C-8 position of the molecule, having undergone reduction of a vinyl group during biosynthesis. Two unrelated classes of 8-vinyl reductase involved in the biosynthesis of chlorophylls are known to exist, BciA and BciB. The genome ofAcaryochloris marinacontains open reading frames (ORFs) encoding proteins displaying high sequence similarity to BciA or BciB, although they are annotated as genes involved in transcriptional control (nmrA) and methanogenesis (frhB), respectively. These genes were introduced into an 8-vinyl chlorophylla-producing ΔbciBstrain ofSynechocystissp. strain PCC 6803, and both were shown to restore synthesis of the pigment with an ethyl group at C-8, demonstrating their activities as 8-vinyl reductases. We propose thatnmrAandfrhBbe reassigned asbciAandbciB, respectively; transcript and proteomic analysis ofAcaryochloris marinareveal that bothbciAandbciBare expressed and their encoded proteins are present in the cell, possibly in order to ensure that all synthesized chlorophyll pigment carries an ethyl group at C-8. Potential reasons for the presence of two 8-vinyl reductases in this strain, which is unique for cyanobacteria, are discussed.IMPORTANCEThe cyanobacteriumAcaryochloris marinais the best-studied phototrophic organism that uses chlorophylldfor photosynthesis. Unique among cyanobacteria sequenced to date, its genome contains ORFs encoding two unrelated enzymes that catalyze the reduction of the C-8 vinyl group of a precursor molecule to an ethyl group. Carrying a reduced C-8 group may be of particular importance to organisms containing chlorophylld. Plant genomes also contain orthologs of both of these genes; thus, the bacterial progenitor of the chloroplast may also have contained bothbciAandbciB.

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