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 Unusually efficient CUG initiation of an overlapping reading frame in POLG mRNA yields novel protein POLGARF

2020 ◽  
Vol 117 (40) ◽  
pp. 24936-24946 ◽  
Author(s):  
Gary Loughran ◽  
Alexander V. Zhdanov ◽  
Maria S. Mikhaylova ◽  
Fedor N. Rozov ◽  
Petr N. Datskevich ◽  
...  
Keyword(s):  
Messenger Rna ◽  
De Novo ◽  
Gene Evolution ◽  
Protein Coding ◽  
Reading Frame ◽  
Serum Stimulation ◽  
Extracellular Signaling ◽  
Functional Investigation ◽  
Mitochondrial Dna Polymerase ◽  
Orf 5

While 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 messenger RNA (mRNA) initiates almost as efficiently (∼60 to 70%) as an AUG in optimal context. This CUG directs translation of a conserved 260-triplet-long overlapping open reading frame (ORF), which we call POLGARF (POLG Alternative Reading Frame). Translation of a short upstream ORF 5′ of this CUG governs the ratio between POLG (the catalytic subunit of mitochondrial DNA polymerase) and POLGARF synthesized from a single POLG mRNA. Functional investigation of POLGARF suggests a role in extracellular signaling. While unprocessed POLGARF localizes to the nucleoli together with its interacting partner C1QBP, serum stimulation results in rapid cleavage and secretion of a POLGARF C-terminal fragment. Phylogenetic analysis shows that POLGARF evolved ∼160 million y ago due to a mammalian-wide interspersed repeat (MIR) transposition into the 5′ leader sequence of the mammalian POLG gene, which became fixed in placental mammals. This discovery of POLGARF unveils a previously undescribed mechanism of de novo protein-coding gene evolution.

scholarly journals A de novo evolved gene in the house mouse regulates female pregnancy cycles

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Chen Xie ◽  
Cemalettin Bekpen ◽  
Sven Künzel ◽  
Maryam Keshavarz ◽  
Rebecca Krebs-Wheaton ◽  
...  
Keyword(s):  
House Mouse ◽  
De Novo ◽  
Specific Protein ◽  
Ribosome Profiling ◽  
Mass Spectrometry Data ◽  
Preimplantation Embryos ◽  
Protein Coding ◽  
Reading Frame ◽  
Protein Coding Genes ◽  
New Genes

The de novo emergence of new genes has been well documented through genomic analyses. However, a functional analysis, especially of very young protein-coding genes, is still largely lacking. Here, we identify a set of house mouse-specific protein-coding genes and assess their translation by ribosome profiling and mass spectrometry data. We functionally analyze one of them, Gm13030, which is specifically expressed in females in the oviduct. The interruption of the reading frame affects the transcriptional network in the oviducts at a specific stage of the estrous cycle. This includes the upregulation of Dcpp genes, which are known to stimulate the growth of preimplantation embryos. As a consequence, knockout females have their second litters after shorter times and have a higher infanticide rate. Given that Gm13030 shows no signs of positive selection, our findings support the hypothesis that a de novo evolved gene can directly adopt a function without much sequence adaptation.

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 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 uORF-Tools – Workflow for the determination of translation-regulatory upstream open reading frames

2018 ◽  
Author(s):  
Anica Scholz ◽  
Florian Eggenhofer ◽  
Rick Gelhausen ◽  
Björn Grüning ◽  
Kathi Zarnack ◽  
...  
Keyword(s):  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Annotation File ◽  
Inhibitory Effects ◽  
Protein Coding ◽  
Reading Frame ◽  
Upstream Open Reading Frames ◽  
Induced Changes ◽  
Reading Frames

AbstractRibosome profiling (ribo-seq) provides a means to analyze active translation by determining ribosome occupancy in a transcriptome-wide manner. The vast majority of ribosome protected fragments (RPFs) resides within the protein-coding sequence of mRNAs. However, commonly reads are also found within the transcript leader sequence (TLS) (aka 5’ untranslated region) preceding the main open reading frame (ORF), indicating the translation of regulatory upstream ORFs (uORFs). Here, we present a workflow for the identification of translation-regulatory uORFs. Specifically, uORF-Tools identifies uORFs within a given dataset and generates a uORF annotation file. In addition, a comprehensive human uORF annotation file, based on 35 ribo-seq files, is provided, which can serve as an alternative input file for the workflow. To assess the translation-regulatory activity of the uORFs, stimulus-induced changes in the ratio of the RPFs residing in the main ORFs relative to those found in the associated uORFs are determined. The resulting output file allows for the easy identification of candidate uORFs, which have translation-inhibitory effects on their associated main ORFs. uORF-Tools is available as a free and open Snakemake workflow at https://github.com/Biochemistry1-FFM/uORF-Tools. It is easily installed and all necessary tools are provided in a version-controlled manner, which also ensures lasting usability. uORF-Tools is designed for intuitive use and requires only limited computing times and resources.

scholarly journals A novel pH-regulated, unusual 603 bp overlapping protein coding gene pop is encoded antisense to ompA in Escherichia coli O157:H7 (EHEC)

2019 ◽  
Author(s):  
Barbara Zehentner ◽  
Zachary Ardern ◽  
Michaela Kreitmeier ◽  
Siegfried Scherer ◽  
Klaus Neuhaus
Keyword(s):  
Antisense Rna ◽  
Prokaryotic Genome ◽  
Antisense Transcription ◽  
Ribosome Profiling ◽  
Escherichia Coli O157 ◽  
Overlapping Genes ◽  
Western Blots ◽  
Protein Coding ◽  
Reading Frame ◽  
Growth Experiments

AbstractAntisense transcription is well known in bacteria. However, translation of antisense RNAs is typically not considered, as the implied overlapping coding at a DNA locus is assumed to be highly improbable. Therefore, such overlapping genes are systematically excluded in prokaryotic genome annotation. Here we report an exceptional 603 bp long open reading frame completely embedded in antisense to the gene of the outer membrane protein ompA. Ribosomal profiling revealed translation of the mRNA and the protein was detected in Western blots. A σ70 promoter, transcription start site, Shine-Dalgarno motif and rho-independent terminator were experimentally validated. A pH-dependent phenotype conferred by the protein was shown in competitive overexpression growth experiments of a translationally arrested mutant versus wild type. We designate this novel gene pop (pH-regulated overlapping protein-coding gene). Increasing evidence based on ribosome-profiling indicates translation of antisense RNA, suggesting that more overlapping genes of unknown function may exist in bacteria.

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 No evidence for phylostratigraphic bias impacting inferences on patterns of gene emergence and evolution

2016 ◽  
Author(s):  
Tomislav Domazet-Lošo ◽  
Anne-Ruxandra Carvunis ◽  
M.Mar Albà ◽  
Martin Sebastijan Šestak ◽  
Robert Bakarić ◽  
...  
Keyword(s):  
De Novo ◽  
Gene Evolution ◽  
False Negative ◽  
False Negative Rate ◽  
Computational Framework ◽  
Protein Coding ◽  
Partial Representation ◽  
Sensitive Tool ◽  
Cast Doubt ◽  
Origin Of Eukaryotes

AbstractPhylostratigraphy is a computational framework for dating the emergence of sequences (usually genes) in a phylogeny. It has been extensively applied to make inferences on patterns of genome evolution, including patterns of disease gene evolution, ontogeny and de novo gene origination. Phylostratigraphy typically relies on BLAST searches along a species tree, but new simulation studies have raised concerns about the ability of BLAST to detect remote homologues and its impact on phylostratigraphic inferences. These simulations called into question some of our previously published work on patterns of gene emergence and evolution inferred from phylostratigraphy. Here, we re-assessed these simulations and found major problems including unrealistic parameter choices, irreproducibility, statistical flaws and partial representation of results. We found that, even with a possible overall BLAST false negative rate between 5-15%, the large majority (>74%) of sequences assigned to a recent evolutionary origin by phylostratigraphy is unaffected by technical concerns about BLAST. Where the results of the simulations did cast doubt on our previous findings, we repeated our analyses but now excluded all questionable sequences. The originally described patterns remained essentially unchanged. These new analyses strongly support our published inferences, including: genes that emerged after the origin of eukaryotes are more likely to be expressed in the ectoderm than in the endoderm or mesoderm in Drosophila, and the de novo emergence of protein-coding genes from non-genic sequences occurs through proto-gene intermediates in yeast. We conclude that BLAST is an appropriate and sufficiently sensitive tool in phylostratigraphic analysis.

scholarly journals Readthrough Errors Purge Deleterious Cryptic Sequences, Facilitating the Birth of Coding Sequences

2020 ◽  
Vol 37 (6) ◽  
pp. 1761-1774 ◽  
Author(s):  
Luke J Kosinski ◽  
Joanna Masel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
De Novo ◽  
Stop Codon ◽  
Spillover Effects ◽  
Structural Disorder ◽  
Ribosome Profiling ◽  
Noncoding Dna ◽  
Protein Coding ◽  
Selection Hypothesis ◽  
Noncoding Sequences

Abstract De novo protein-coding innovations sometimes emerge from ancestrally noncoding DNA, despite the expectation that translating random sequences is overwhelmingly likely to be deleterious. The “preadapting selection” hypothesis claims that emergence is facilitated by prior, low-level translation of noncoding sequences via molecular errors. It predicts that selection on polypeptides translated only in error is strong enough to matter and is strongest when erroneous expression is high. To test this hypothesis, we examined noncoding sequences located downstream of stop codons (i.e., those potentially translated by readthrough errors) in Saccharomyces cerevisiae genes. We identified a class of “fragile” proteins under strong selection to reduce readthrough, which are unlikely substrates for co-option. Among the remainder, sequences showing evidence of readthrough translation, as assessed by ribosome profiling, encoded C-terminal extensions with higher intrinsic structural disorder, supporting the preadapting selection hypothesis. The cryptic sequences beyond the stop codon, rather than spillover effects from the regular C-termini, are primarily responsible for the higher disorder. Results are robust to controlling for the fact that stronger selection also reduces the length of C-terminal extensions. These findings indicate that selection acts on 3′ UTRs in Saccharomyces cerevisiae to purge potentially deleterious variants of cryptic polypeptides, acting more strongly in genes that experience more readthrough errors.

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.

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