scholarly journals Evidence for functional and non-functional classes of peptides translated from long non-coding RNAs

2016 ◽  
Author(s):  
Jorge Ruiz-Orera ◽  
Pol Verdaguer-Grau ◽  
José Luis Villanueva-Cañas ◽  
Xavier Messeguer ◽  
M Mar Albà
Keyword(s):  
De Novo ◽  
Gene Evolution ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Polymorphism Analysis ◽  
Proteomics Data ◽  
Functional Protein ◽  
Codon Composition ◽  
Functional Classes ◽  
Chance Occurrence

AbstractThere is accumulating evidence that some genes have originated de novo from previously non-coding genomic sequences. However, the processes underlying de novo gene birth are still enigmatic. In particular, the appearance of a new functional protein seems highly improbable unless there is already a pool of neutrally evolving peptides that can at some point acquire new functions. Here we show for the first time that such peptides do not only exist but that they are prevalent among the translation products of mouse genes that lack homologues in rat and human. The data suggests that the translation of these peptides is due to the chance occurrence of open reading frames with a favorable codon composition. Our approach combines ribosome profiling experiments, proteomics data and non-synonymous and synonymous nucleotide polymorphism analysis. We propose that effectively neutral processes involving the expression of thousands of transcripts all the way down to proteins provide a basis for de novo gene evolution.

scholarly journals An Improved Human smORF Annnotation Workflow Combining De Novo Transcriptome Assembly and Ribo-Seq

2019 ◽  
Author(s):  
Thomas F. Martinez ◽  
Qian Chu ◽  
Cynthia Donaldson ◽  
Dan Tan ◽  
Maxim N. Shokhirev ◽  
...  
Keyword(s):  
De Novo ◽  
Transcriptome Assembly ◽  
Human Leukocyte ◽  
Open Reading Frames ◽  
Translation Efficiency ◽  
Proteomics Data ◽  
Protein Coding ◽  
Leukocyte Antigen ◽  
Human Genes ◽  
Small Open Reading Frames

Protein-coding small open reading frames (smORFs) are emerging as an important class of genes, however, the coding capacity of smORFs in the human genome is unclear. By integrating de novo transcriptome assembly and Ribo-Seq, we confidently annotate thousands of novel translated smORFs in three human cell lines. We find that smORF translation prediction is noisier than for annotated coding sequences, underscoring the importance of analyzing multiple experiments and footprinting conditions. These smORFs are located within non-coding and antisense transcripts, the UTRs of mRNAs, and unannotated transcripts. Analysis of RNA levels and translation efficiency during cellular stress identifies regulated smORFs, providing an approach to select smORFs for further investigation. Sequence conservation and signatures of positive selection indicate that encoded microproteins are likely functional. Additionally, proteomics data from enriched human leukocyte antigen complexes validates the translation of hundreds of smORFs and positions them as a source of novel antigens. Thus, smORFs represent a significant number of important, yet unexplored human genes.

scholarly journals A spectral analysis approach to detect actively translated open reading frames in high-resolution ribosome profiling data

2015 ◽  
Author(s):  
Lorenzo Calviello ◽  
Neelanjan Mukherjee ◽  
Emanuel Wyler ◽  
Henrik Zauber ◽  
Antje Hirsekorn ◽  
...  
Keyword(s):  
Spectral Analysis ◽  
Gene Expression Regulation ◽  
De Novo ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Mass Spectrometry Data ◽  
Hek293 Cells ◽  
Protein Coding ◽  
Reading Frame ◽  
Reading Frames

RNA sequencing protocols allow for quantifying gene expression regulation at each individual step, from transcription to protein synthesis. Ribosome Profiling (Ribo-seq) maps the positions of translating ribosomes over the entire transcriptome. Despite its great potential, a rigorous statistical approach to identify translated regions by means of the characteristic three-nucleotide periodicity of Ribo-seq data is not yet available. To fill this gap, we developed RiboTaper, which quantifies the significance of periodic Ribo-seq reads via spectral analysis methods. We applied RiboTaper on newly generated, deep Ribo-seq data in HEK293 cells, to derive an extensive map of translation that covers Open Reading Frame (ORF) annotations for more than 11,000 protein- coding genes. We also find distinct ribosomal signatures for several hundred detected upstream ORFs and ORFs in annotated non-coding genes (ncORFs). Mass spectrometry data confirms that RiboTaper achieves excellent coverage of the cellular proteome and validates dozens of novel peptide products. Collectively, RiboTaper (available at https://ohlerlab.mdc-berlin.de/software/ ) is a powerful method for comprehensive de novo identification of actively used ORFs in the human genome.

scholarly journals Are Antisense Proteins in Prokaryotes Functional?

2020 ◽  
Author(s):  
Zachary Ardern ◽  
Klaus Neuhaus ◽  
Siegfried Scherer
Keyword(s):  
Gene Evolution ◽  
Bacterial Species ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Evolutionary Analysis ◽  
Protein Coding ◽  
Novel Proteins ◽  
Show Evidence ◽  
Exciting Potential ◽  
Genome Annotations

AbstractMany prokaryotic RNAs are transcribed from loci outside of annotated protein coding genes. Across bacterial species hundreds of short open reading frames antisense to annotated genes show evidence of both transcription and translation, for instance in ribosome profiling data. Determining the functional fraction of these protein products awaits further research, including insights from studies of molecular interactions and detailed evolutionary analysis. There are multiple lines of evidence however that many of these newly discovered proteins are of use to the organism. Condition-specific phenotypes have been characterised for a few. These proteins should be added to genome annotations, and the methods for predicting them standardised. Evolutionary analysis of these typically young sequences also may provide important insights into gene evolution. This research should be prioritised for its exciting potential to uncover large numbers of novel proteins with extremely diverse potential practical uses, including applications in synthetic biology and responding to pathogens.

scholarly journals A Coding Sequence-Embedded Principle Governs Translational Reading Frame Fidelity

Research ◽  
2018 ◽  
Vol 2018 ◽  
pp. 1-15 ◽  
Author(s):  
Ji Wan ◽  
Xiangwei Gao ◽  
Yuanhui Mao ◽  
Xingqian Zhang ◽  
Shu-Bing Qian
Keyword(s):  
18S Rrna ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Start Codon ◽  
Data Sets ◽  
Protein Coding ◽  
Reading Frame ◽  
Codon Composition ◽  
Sequence Elements ◽  
Correct Reading

Upon initiation at a start codon, the ribosome must maintain the correct reading frame for hundreds of codons in order to produce functional proteins. While some sequence elements are able to trigger programmed ribosomal frameshifting (PRF), very little is known about how the ribosome normally prevents spontaneous frameshift errors that can have dire consequences if uncorrected. Using high resolution ribosome profiling data sets, we discovered that the translating ribosome uses the 3′ end of 18S rRNA to scan the AUG-like codons after the decoding process. The postdecoding mRNA:rRNA interaction not only contributes to predominant translational pausing, but also provides a retrospective mechanism to safeguard the ribosome in the correct reading frame. Partially eliminating the AUG-like “sticky” codons in the reporter message leads to increased +1 frameshift errors. Remarkably, mutating the highly conserved CAU triplet of 18S rRNA globally changes the codon “stickiness”. Further supporting the role of “sticky” sequences in reading frame maintenance, the codon composition of open reading frames is highly optimized across eukaryotic genomes. These results suggest an important layer of information embedded within the protein-coding sequences that instructs the ribosome to ensure reading frame fidelity during translation.

scholarly journals De novo gene evolution: How do we transition from non-coding to coding?

2017 ◽  
Author(s):  
Jorge Ruiz-Orera ◽  
José Luis Villanueva-Cañas ◽  
William Blevins ◽  
M.Mar Albà
Keyword(s):  
De Novo ◽  
Gene Evolution ◽  
Neutral Evolution ◽  
Functional Protein ◽  
Protein Coding ◽  
Coding Sequences ◽  
Sequence Composition ◽  
Protein Coding Genes ◽  
Small Proteins ◽  
De Novo Gene

Recent years have witnessed the discovery of protein–coding genes which appear to have evolved de novo from previously non-coding sequences. This has changed the long-standing view that coding sequences can only evolve from other coding sequences. However, there are still many open questions regarding how new protein-coding sequences can arise from non-genic DNA. Two prerequisites for the birth of a new functional protein-coding gene are that the corresponding DNA fragment is transcribed and that it is also translated. Transcription is known to be pervasive in the genome, producing a large number of transcripts that do not correspond to conserved protein-coding genes, and which are usually annotated as long non-coding RNAs (lncRNA). Recently, sequencing of ribosome protected fragments (Ribo-Seq) has provided evidence that many of these transcripts actually translate small proteins. We have used mouse non-synonymous and synonymous variation data to estimate the strength of purifying selection acting on the translated open reading frames (ORFs). Whereas a subset of the lncRNAs are likely to actually be true protein-coding genes (and thus previously misclassified), the bulk of lncRNAs code for proteins which show variation patterns consistent with neutral evolution. We also show that the ORFs that have a more favorable, coding-like, sequence composition are more likely to be translated than other ORFs in lncRNAs. This study provides strong evidence that there is a large and ever-changing reservoir of lowly abundant proteins; some of these peptides may become useful and act as seeds for de novo gene evolution.

scholarly journals Unprecedentedly efficient CUG initiation of an overlapping reading frame in POLG mRNA yields novel protein POLGARF

2020 ◽  
Author(s):  
G Loughran ◽  
AV Zhdanov ◽  
MS Mikhaylova ◽  
FN Rozov ◽  
PN Datskevich ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
De Novo ◽  
Gene Evolution ◽  
Ribosome Profiling ◽  
Dual Coding ◽  
Protein Coding ◽  
Reading Frame ◽  
Serum Stimulation ◽  
Functional Investigation ◽  
Mitochondrial Dna Polymerase

AbstractWhile near cognate codons are frequently used for translation initiation in eukaryotes, their efficiencies are usually low (<10% compared to an AUG in optimal context). Here we describe a rare case of highly efficient near cognate initiation. A CUG triplet located in the 5’ leader of POLG mRNA initiates almost as efficiently (~60-70%) as an AUG in optimal context. This CUG directs translation of a conserved 260 triplet-long overlapping ORF, which we call POLGARF (POLGAlternative Reading Frame). Translation of a short upstream ORF 5’ of this CUG governs the ratio between DNA polymerase and POLGARF produced from a single POLG mRNA. Functional investigation of POLGARF points to extracellular signalling. While unprocessed POLGARF resides in the nucleoli together with its interacting partner C1QBP, serum stimulation results in rapid secretion of POLGARF C-terminal fragment. Phylogenetic analysis shows that POLGARF evolved ~160 million years ago due to an MIR transposition into the 5’ leader sequence of the mammalian POLG gene which became fixed in placental mammals. The discovery of POLGARF unveils a previously undescribed mechanism of de novo protein-coding gene evolution.Significance StatementIn this study, we describe previously unknown mechanism of de novo protein-coding gene evolution. We show that the POLG gene, which encodes the catalytic subunit of mitochondrial DNA polymerase, is in fact a dual coding gene. Ribosome profiling, phylogenetic conservation, and reporter construct analyses all demonstrate that POLG mRNA possesses a conserved CUG codon which serves as a start of translation for an exceptionally long overlapping open reading frame (260 codons in human) present in all placental mammals. We called the protein encoded in this alternative reading frame POLGARF. We provide evidence that the evolution of POLGARF was incepted upon insertion of an MIR transposable element of the SINE family.

scholarly journals REPARATION: Ribosome Profiling Assisted (Re-)Annotation of Bacterial genomes

2017 ◽  
Author(s):  
Elvis Ndah ◽  
Veronique Jonckheere ◽  
Adam Giess ◽  
Eivind Valen ◽  
Gerben Menschaert ◽  
...  
Keyword(s):  
Genome Annotation ◽  
De Novo ◽  
Bacterial Species ◽  
Prokaryotic Genome ◽  
Protein Translation ◽  
Ribosome Profiling ◽  
Proteomics Data ◽  
Bacterial Genomes ◽  
Sequence Context ◽  
Automated Methods

ABSTRACTProkaryotic genome annotation is highly dependent on automated methods, as manual curation cannot keep up with the exponential growth of sequenced genomes. Current automated methods depend heavily on sequence context and often underestimate the complexity of the proteome. We developed REPARATION (RibosomeE Profiling Assisted (Re-)AnnotaTION), a de novo algorithm that takes advantage of experimental protein translation evidence from ribosome profiling (Ribo-seq) to delineate translated open reading frames (ORFs) in bacteria, independent of genome annotation. REPARATION evaluates all possible ORFs in the genome and estimates minimum thresholds based on a growth curve model to screen for spurious ORFs. We applied REPARATION to three annotated bacterial species to obtain a more comprehensive mapping of their translation landscape in support of experimental data. In all cases, we identified hundreds of novel (small) ORFs including variants of previously annotated ORFs. Our predictions were supported by matching mass spectrometry (MS) proteomics data, sequence composition and conservation analysis. REPARATION is unique in that it makes use of experimental translation evidence to perform de novo ORF delineation in bacterial genomes irrespective of the sequence context of the reading frame.

Translational Landscape of Protein-Coding and Non-Protein-Coding RNAs upon Light Exposure in Arabidopsis

2019 ◽  
Vol 61 (3) ◽  
pp. 536-545 ◽  
Author(s):  
Yukio Kurihara ◽  
Yuko Makita ◽  
Haruka Shimohira ◽  
Tomoya Fujita ◽  
Shintaro Iwasaki ◽  
...  
Keyword(s):  
Blue Light ◽  
Environmental Changes ◽  
De Novo ◽  
Light Exposure ◽  
Monochromatic Light ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Translation Efficiency ◽  
Protein Coding ◽  
Negative Effect

Abstract Light is one of the most essential environmental clues for plant growth and morphogenesis. Exposure to blue monochromatic light from darkness is a turning point for plant biological activity, and as a result dramatic changes in gene expression occur. To understand the translational impacts of blue light, we have performed ribosome profiling analysis and called translated open reading frames (ORFs) de novo within not only mRNAs but also non-coding RNAs (ncRNAs). Translation efficiency of 3,823 protein-coding ORFs, such as nuclear chloroplast-related genes, was up-regulated by blue light exposure. Moreover, the translational activation of the microRNA biogenesis-related genes, DCL1 and HYL1, was induced by blue light. Considering the 3-nucleotide codon periodicity of ribosome footprints, a few hundred short ORFs lying on ncRNAs and upstream ORFs (uORFs) on mRNAs were found that had differential translation status between blue light and dark. uORFs are known to have a negative effect on the expression of the main ORFs (mORFs) on the same mRNAs. Our analysis suggests that the translation of uORFs is likely to be more stimulated than that of the corresponding mORFs, and uORF-mediated translational repression of the mORFs in five genes was alleviated by blue light exposure. With data-based annotation of the ORFs, our analysis provides insights into the translatome in response to environmental changes, such as those involving light.

scholarly journals Enhancers Facilitate the Birth of De Novo Genes and Gene Integration into Regulatory Networks

2019 ◽  
Vol 37 (4) ◽  
pp. 1165-1178 ◽  
Author(s):  
Paco Majic ◽  
Joshua L Payne
Keyword(s):  
Gene Expression ◽  
Regulatory Networks ◽  
De Novo ◽  
Gene Evolution ◽  
Mammalian Species ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Open Reading Frames ◽  
Open Chromatin ◽  
Reading Frames

Abstract Regulatory networks control the spatiotemporal gene expression patterns that give rise to and define the individual cell types of multicellular organisms. In eumetazoa, distal regulatory elements called enhancers play a key role in determining the structure of such networks, particularly the wiring diagram of “who regulates whom.” Mutations that affect enhancer activity can therefore rewire regulatory networks, potentially causing adaptive changes in gene expression. Here, we use whole-tissue and single-cell transcriptomic and chromatin accessibility data from mouse to show that enhancers play an additional role in the evolution of regulatory networks: They facilitate network growth by creating transcriptionally active regions of open chromatin that are conducive to de novo gene evolution. Specifically, our comparative transcriptomic analysis with three other mammalian species shows that young, mouse-specific intergenic open reading frames are preferentially located near enhancers, whereas older open reading frames are not. Mouse-specific intergenic open reading frames that are proximal to enhancers are more highly and stably transcribed than those that are not proximal to enhancers or promoters, and they are transcribed in a limited diversity of cellular contexts. Furthermore, we report several instances of mouse-specific intergenic open reading frames proximal to promoters showing evidence of being repurposed enhancers. We also show that open reading frames gradually acquire interactions with enhancers over macroevolutionary timescales, helping integrate genes—those that have arisen de novo or by other means—into existing regulatory networks. Taken together, our results highlight a dual role of enhancers in expanding and rewiring gene regulatory networks.

scholarly journals Kozak sequence acts as a negative regulator for de novo transcription initiation of newborn coding sequences in the plant genome

2020 ◽  
Author(s):  
Takayuki Hata ◽  
Soichirou Satoh ◽  
Naoto Takada ◽  
Mitsuhiro Matsuo ◽  
Junichi Obokata
Keyword(s):  
Transcription Initiation ◽  
Cultured Cells ◽  
De Novo ◽  
Gene Evolution ◽  
Firefly Luciferase ◽  
Open Reading Frames ◽  
Negative Regulator ◽  
Plant Genome ◽  
Coding Sequences ◽  
Precise Position

ABSTRACTThe manner in which newborn coding sequences and their transcriptional competency emerge during the process of gene evolution remains unclear. Here, we experimentally simulated eukaryotic gene origination processes by mimicking horizontal gene transfer events in the plant genome. We mapped the precise position of the transcription start sites (TSSs) of hundreds of newly introduced promoterless firefly luciferase (LUC) coding sequences in the genome of Arabidopsis thaliana cultured cells. The systematic characterization of the LUC-TSSs revealed that 80% of them occurred under the influence of endogenous promoters, while the remainder underwent de novo activation in the intergenic regions, starting from pyrimidine-purine dinucleotides. These de novo TSSs obeyed unexpected rules; they predominantly occurred ~100 bp upstream of the LUC inserts and did not overlap with Kozak-containing putative open reading frames (ORFs). These features were the output of the immediate responses to the sequence insertions, rather than a bias in the screening of the LUC gene function. Regarding the wild-type genic TSSs, they appeared to have evolved to lack any ORFs in their vicinities. Therefore, the repulsion by the de novo TSSs of Kozak-containing ORFs described above might be the first selection gate for the occurrence and evolution of TSSs in the plant genome. Based on these results, we characterized the de novo type of TSS identified in the plant genome and discuss its significance in genome evolution.

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