Global Transcriptome Characterization and Assembly of the Thermophilic Ascomycete Chaetomium thermophilum

A correct genome annotation is fundamental for research in the field of molecular and structural biology. The annotation of the reference genome of Chaetomium thermophilum has been reported previously, but it is essentially limited to open reading frames (ORFs) of protein coding genes and contains only a few noncoding transcripts. In this study, we identified and annotated full-length transcripts of C. thermophilum by deep RNA sequencing. We annotated 7044 coding genes and 4567 noncoding genes. Astonishingly, 23% of the coding genes are alternatively spliced. We identified 679 novel coding genes as well as 2878 novel noncoding genes and corrected the structural organization of more than 50% of the previously annotated genes. Furthermore, we substantially extended the Gene Ontology (GO) and Enzyme Commission (EC) lists, which provide comprehensive search tools for potential industrial applications and basic research. The identified novel transcripts and improved annotation will help to understand the gene regulatory landscape in C. thermophilum. The analysis pipeline developed here can be used to build transcriptome assemblies and identify coding and noncoding RNAs of other species.

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Global Transcriptome Characterization and Assembly of Thermophilic Ascomycete Chaetomium thermophilum

10.1101/826354 ◽

2019 ◽

Author(s):

Amit Singh ◽

Géza Schermann ◽

Sven Reislöhner ◽

Nikola Kellner ◽

Ed Hurt ◽

...

Keyword(s):

Basic Research ◽

Industrial Applications ◽

Open Reading Frames ◽

Annotation Database ◽

Go Annotation ◽

Chaetomium Thermophilum ◽

Alternatively Spliced ◽

Comprehensive Search ◽

R Packages ◽

Novel Transcripts

A correct genome annotation is fundamental for research in the field of molecular and structural biology. The annotation of the reference genome Chaetomium thermophilum has been reported previously, but it is limited to open reading frames (ORFs) of genes and contains only a few noncoding transcripts. In this study, we identified and annotated by deep RNA sequencing full-length transcripts of C.thermophilum. We annotated 7044 coding genes and a large number of noncoding genes (n=4567). Astonishingly, 23% of the coding genes are alternatively spliced. We identified 679 novel coding genes and corrected the structural organization of more than 50% of the previously annotated genes. Furthermore, we substantially extended the Gene Ontology (GO) and Enzyme Commission (EC) lists, which provide comprehensive search tools for potential industrial applications and basic research. The identified novel transcripts and improved annotation will help understanding the gene regulatory landscape in C.thermophilum. The analysis pipeline developed here can be used to build transcriptome assemblies and identify coding and noncoding RNAs of other species. The R packages for gene and GO annotation database can be found under https://www.bzh.uni-heidelberg.de/brunner/Chaetomium_thermophilum.

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Long-read cDNA sequencing identifies functional pseudogenes in the human transcriptome

Genome Biology ◽

10.1186/s13059-021-02369-0 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Robin-Lee Troskie ◽

Yohaann Jafrani ◽

Tim R. Mercer ◽

Adam D. Ewing ◽

Geoffrey J. Faulkner ◽

...

Keyword(s):

Cultured Cells ◽

Open Reading Frames ◽

Cdna Sequencing ◽

Protein Coding ◽

Dynamic Component ◽

Gene Copies ◽

Long Read ◽

Normal Human ◽

Reading Frames ◽

Transcriptional Landscape

AbstractPseudogenes are gene copies presumed to mainly be functionless relics of evolution due to acquired deleterious mutations or transcriptional silencing. Using deep full-length PacBio cDNA sequencing of normal human tissues and cancer cell lines, we identify here hundreds of novel transcribed pseudogenes expressed in tissue-specific patterns. Some pseudogene transcripts have intact open reading frames and are translated in cultured cells, representing unannotated protein-coding genes. To assess the biological impact of noncoding pseudogenes, we CRISPR-Cas9 delete the nucleus-enriched pseudogene PDCL3P4 and observe hundreds of perturbed genes. This study highlights pseudogenes as a complex and dynamic component of the human transcriptional landscape.

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Disrupting upstream translation in mRNAs is associated with human disease

Nature Communications ◽

10.1038/s41467-021-21812-1 ◽

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.

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Overexpression-based detection of translatable circular RNAs is vulnerable to coexistent linear RNA byproducts

10.1101/2021.03.23.433163 ◽

2021 ◽

Author(s):

Yanyi Jiang ◽

Xiaofan Chen ◽

Wei Zhang

Keyword(s):

Open Reading Frames ◽

Systematic Evaluation ◽

Circular Rnas ◽

Protein Coding ◽

Rolling Circle ◽

Functional Investigation ◽

Overexpression System ◽

Translation Signals ◽

Coding Potential ◽

Reading Frames

AbstractIn RNA field, the demarcation between coding and non-coding has been negotiated by the recent discovery of occasionally translated circular RNAs (circRNAs). Although absent of 5’ cap structure, circRNAs can be translated cap-independently. Complementary intron-mediated overexpression is one of the most utilized methodologies for circRNA research but not without bearing echoing skepticism for its poorly defined mechanism and latent coexistent side products. In this study, leveraging such circRNA overexpression system, we have interrogated the protein-coding potential of 30 human circRNAs containing infinite open reading frames in HEK293T cells. Surprisingly, pervasive translation signals are detected by immunoblotting. However, intensive mutagenesis reveals that numerous translation signals are generated independently of circRNA synthesis. We have developed a dual tag strategy to isolate translation noise and directly demonstrate that the fallacious translation signals originate from cryptically spliced linear transcripts. The concomitant linear RNA byproducts, presumably concatemers, can be translated to allow pseudo rolling circle translation signals, and can involve backsplicing junction (BSJ) to disqualify the BSJ-based evidence for circRNA translation. We also find non-AUG start codons may engage in the translation initiation of circRNAs. Taken together, our systematic evaluation sheds light on heterogeneous translational outputs from circRNA overexpression vector and comes with a caveat that ectopic overexpression technique necessitates extremely rigorous control setup in circRNA translation and functional investigation.

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A micropeptide encoded by lncRNA MIR155HG suppresses autoimmune inflammation via modulating antigen presentation

Science Advances ◽

10.1126/sciadv.aaz2059 ◽

2020 ◽

Vol 6 (21) ◽

pp. eaaz2059 ◽

Cited By ~ 4

Author(s):

Liman Niu ◽

Fangzhou Lou ◽

Yang Sun ◽

Libo Sun ◽

Xiaojie Cai ◽

...

Keyword(s):

Antigen Presentation ◽

Inflammatory Diseases ◽

Open Reading Frames ◽

Protein Coding ◽

Histocompatibility Complex ◽

Antigen Trafficking ◽

Heat Shock Cognate Protein ◽

Antigen Presenting ◽

Cognate Protein ◽

Reading Frames

Many annotated long noncoding RNAs (lncRNAs) harbor predicted short open reading frames (sORFs), but the coding capacities of these sORFs and the functions of the resulting micropeptides remain elusive. Here, we report that human lncRNA MIR155HG encodes a 17–amino acid micropeptide, which we termed miPEP155 (P155). MIR155HG is highly expressed by inflamed antigen-presenting cells, leading to the discovery that P155 interacts with the adenosine 5′-triphosphate binding domain of heat shock cognate protein 70 (HSC70), a chaperone required for antigen trafficking and presentation in dendritic cells (DCs). P155 modulates major histocompatibility complex class II–mediated antigen presentation and T cell priming by disrupting the HSC70-HSP90 machinery. Exogenously injected P155 improves two classical mouse models of DC-driven auto inflammation. Collectively, we demonstrate the endogenous existence of a micropeptide encoded by a transcript annotated as “non-protein coding” and characterize a micropeptide as a regulator of antigen presentation and a suppressor of inflammatory diseases.

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When Long Noncoding Becomes Protein Coding

Molecular and Cellular Biology ◽

10.1128/mcb.00528-19 ◽

2020 ◽

Vol 40 (6) ◽

Cited By ~ 14

Author(s):

Corrine Corrina R. Hartford ◽

Ashish Lal

Keyword(s):

Cell Division ◽

Cell Signaling ◽

Transcription Regulation ◽

Noncoding Rnas ◽

Long Noncoding Rnas ◽

Open Reading Frames ◽

Protein Coding ◽

Small Proteins ◽

Coding Potential ◽

Reading Frames

ABSTRACT Recent advancements in genetic and proteomic technologies have revealed that more of the genome encodes proteins than originally thought possible. Specifically, some putative long noncoding RNAs (lncRNAs) have been misannotated as noncoding. Numerous lncRNAs have been found to contain short open reading frames (sORFs) which have been overlooked because of their small size. Many of these sORFs encode small proteins or micropeptides with fundamental biological importance. These micropeptides can aid in diverse processes, including cell division, transcription regulation, and cell signaling. Here we discuss strategies for establishing the coding potential of putative lncRNAs and describe various functions of known micropeptides.

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Powdery Mildew-Induced Mla mRNAs Are Alternatively Spliced and Contain Multiple Upstream Open Reading Frames

PLANT PHYSIOLOGY ◽

10.1104/pp.014407 ◽

2003 ◽

Vol 131 (2) ◽

pp. 558-567 ◽

Cited By ~ 98

Author(s):

Dennis A. Halterman ◽

Fusheng Wei ◽

Roger P. Wise

Keyword(s):

Powdery Mildew ◽

Open Reading Frames ◽

Upstream Open Reading Frames ◽

Alternatively Spliced ◽

Reading Frames

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Identification of Proteins Associated with Murine Cytomegalovirus Virions

Journal of Virology ◽

10.1128/jvi.78.20.11187-11197.2004 ◽

2004 ◽

Vol 78 (20) ◽

pp. 11187-11197 ◽

Cited By ~ 105

Author(s):

Lisa M. Kattenhorn ◽

Ryan Mills ◽

Markus Wagner ◽

Alexandre Lomsadze ◽

Vsevolod Makeev ◽

...

Keyword(s):

Gene Prediction ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Open Reading Frames ◽

Murine Cytomegalovirus ◽

Prediction Algorithm ◽

Sequencing Analysis ◽

Protein Coding ◽

Coding Potential ◽

Reading Frames

ABSTRACT Proteins associated with the murine cytomegalovirus (MCMV) viral particle were identified by a combined approach of proteomic and genomic methods. Purified MCMV virions were dissociated by complete denaturation and subjected to either separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in-gel digestion or treated directly by in-solution tryptic digestion. Peptides were separated by nanoflow liquid chromatography and analyzed by tandem mass spectrometry (LC-MS/MS). The MS/MS spectra obtained were searched against a database of MCMV open reading frames (ORFs) predicted to be protein coding by an MCMV-specific version of the gene prediction algorithm GeneMarkS. We identified 38 proteins from the capsid, tegument, glycoprotein, replication, and immunomodulatory protein families, as well as 20 genes of unknown function. Observed irregularities in coding potential suggested possible sequence errors in the 3′-proximal ends of m20 and M31. These errors were experimentally confirmed by sequencing analysis. The MS data further indicated the presence of peptides derived from the unannotated ORFs ORFc225441-226898 (m166.5) and ORF105932-106072. Immunoblot experiments confirmed expression of m166.5 during viral infection.

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Long antiparallel open reading frames are unlikely to be encoding essential proteins in prokaryotic genomes

10.1101/724807 ◽

2019 ◽

Author(s):

Denis Moshensky ◽

Andrei Alexeevski

Keyword(s):

Negative Selection ◽

Stop Codon ◽

Biological Significance ◽

Open Reading Frames ◽

Overlapping Genes ◽

Base Pairs ◽

Protein Coding ◽

Essential Proteins ◽

Prokaryotic Genomes ◽

Reading Frames

AbstractThe origin and evolution of genes that have common base pairs (overlapping genes) are of particular interest due to their influencing each other. Especially intriguing are gene pairs with long overlaps. In prokaryotes, co-directional overlaps longer than 60 bp were shown to be nonexistent except for some instances. A few antiparallel prokaryotic genes with long overlaps were described in the literature. We have analyzed putative long antiparallel overlapping genes to determine whether open reading frames (ORFs) located opposite to genes (antiparallel ORFs) can be protein-coding genes.We have confirmed that long antiparallel ORFs (AORFs) are observed reliably to be more frequent than expected. There are 10 472 000 AORFs in 929 analyzed genomes with overlap length more than 180 bp. Stop codons on the opposite to the coding strand are avoided in 2 898 cases with Benjamini-Hochberg threshold 0.01.Using Ka/Ks ratio calculations, we have revealed that long AORFs do not affect the type of selection acting on genes in a vast majority of cases. This observation indicates that long AORFs translations commonly are not under negative selection.The demonstrative example is 282 longer than 1 800 bp AORFs found opposite to extremely conserved dnaK genes. Translations of these AORFs were annotated “glutamate dehydrogenases” and were included into Pfam database as third protein family of glutamate dehydrogenases, PF10712. Ka/Ks analysis has demonstrated that if these translations correspond to proteins, they are not subjected by negative selection while dnaK genes are under strong stabilizing selection. Moreover, we have found other arguments against the hypothesis that these AORFs encode essential proteins, proteins indispensable for cellular machinery.However, some AORFs, in particular, dnaK related, have been found slightly resisting to synonymous changes in genes. It indicates the possibility of their translation. We speculate that translations of certain AORFs might have a functional role other than encoding essential proteins.Essential genes are unlikely to be encoded by AORFs in prokaryotic genomes. Nevertheless, some AORFs might have biological significance associated with their translations.Author summaryGenes that have common base pairs are called overlapping genes. We have examined the most intriguing case: if gene pairs encoded on opposite DNA strands exist in prokaryotes. An intersection length threshold 180 bp has been used. A few such pairs of genes were experimentally confirmed.We have detected all long antiparallel ORFs in 929 prokaryotic genomes and have found that the number of open reading frames, located opposite to annotated genes, is much more than expected according to statistical model. We have developed a measure of stop codon avoidance on the opposite strand. The lengths of found antiparallel ORFs with stop codon avoidance are typical for prokaryotic genes.Comparative genomics analysis shows that long antiparallel ORFs (AORFs) are unlikely to be essential protein-coding genes. We have analyzed distributions of features typical for essential proteins among formal translations of all long AORFs: prevalence of negative selection, non-uniformity of a conserved positions distribution in a multiple alignment of homologous proteins, the character of homologs distribution in phylogenetic tree of prokaryotes. All of them have not been observed for the majority of long AORFs. Particularly, the same results have been obtained for some experimentally confirmed AOGs.Thus, pairs of antiparallel overlapping essential genes are unlikely to exist. On the other hand, some antiparallel ORFs affect the evolution of genes opposite that they are located. Consequently, translations of some antiparallel ORFs might have yet unknown biological significance.

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