Noncoding AUG circRNAs constitute an abundant and conserved subclass of circles

Circular RNAs (circRNAs) are a subset of noncoding RNAs previously considered as products of missplicing. Now, circRNAs are considered functional molecules, although to date, only few functions have been experimentally validated. Here, based on RNA sequencing from the ENCODE consortium, we identify and characterize a subset of circRNAs, coined AUG circRNAs, encompassing the annotated translational start codon from the protein-coding host genes. AUG circRNAs are more abundantly expressed and conserved than other groups of circRNAs, and they display flanking sequences that suggest an Alu-independent mechanism of biogenesis. The AUG circRNAs contain part of bona fide open reading frame, and in the recent years, several studies have reported cases of circRNA translation. However, using thorough cross-species analysis, extensive ribosome profiling, proteomics analyses, and experimental data on a selected panel of AUG circRNAs, we observe no indications of translation of AUG circRNAs or any other circRNAs. Our data provide a comprehensive classification of circRNAs and, collectively, the data suggest that the AUG circRNAs constitute an abundant subclass of circRNAs produced independently of primate-specific Alu elements.

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Non-coding AUG circRNAs constitute an abundant and conserved subclass of circles

10.1101/328104 ◽

2018 ◽

Cited By ~ 1

Author(s):

Lotte VW Stagsted ◽

Katrine M Nielsen ◽

Iben Daugaard ◽

Thomas B Hansen

Keyword(s):

Species Conservation ◽

Ribosome Profiling ◽

Start Codon ◽

Circular Rnas ◽

Protein Coding ◽

Species Analysis ◽

Bona Fide ◽

Translational Start Codon ◽

Comprehensive Classification ◽

Translational Start

AbstractCircular RNAs (circRNAs) are a subset of non-coding RNAs (ncRNAs) previously considered as products of missplicing. Now, circRNAs are considered functional molecules, although to date, only few functions have been experimentally validated, and therefore the vast majority of circRNAs are without known relevance. Here, based on RNA sequencing from the ENCODE consortium, we identify and characterize a subset of circRNAs, coined AUG circRNAs, encompassing the annotated translational start codon from the protein-coding host genes. AUG circRNAs are more abundantly expressed and conserved than other groups of circRNAs, and they display an Alu-independent mechanism of biogenesis. The AUG circRNAs contain part of bona fide ORF, and in the recent years, several studies have reported cases of circRNA translation. However, using thorough cross-species analysis, extensive ribosome profiling, proteomics analyses, and experimental data on a selected panel of AUG circRNAs, we observe no indications of translation of AUG circRNAs or any other circRNAs. Our data provide a comprehensive classification of circRNAs and, collectively, the data suggest that the AUG circRNAs constitute an abundant subclass of circRNAs produced independently of primate-specific Alu elements. Moreover, AUG circRNAs exhibit high cross-species conservation and are therefore likely to be functionally relevant.

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A Coding Sequence-Embedded Principle Governs Translational Reading Frame Fidelity

Research ◽

10.1155/2018/7089174 ◽

2018 ◽

Vol 2018 ◽

pp. 1-15 ◽

Cited By ~ 1

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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Single-molecule FRET studies on the cotranscriptional folding of a thiamine pyrophosphate riboswitch

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1712983115 ◽

2017 ◽

Vol 115 (2) ◽

pp. 331-336 ◽

Cited By ~ 19

Author(s):

Heesoo Uhm ◽

Wooyoung Kang ◽

Kook Sun Ha ◽

Changwon Kang ◽

Sungchul Hohng

Keyword(s):

Single Molecule ◽

Start Codon ◽

Thiamine Pyrophosphate ◽

Controlled Study ◽

Fluorescence Studies ◽

Physical Mechanisms ◽

Single Molecule Fret ◽

Transcriptional Pausing ◽

Translational Start Codon ◽

Translational Start

Because RNAs fold as they are being synthesized, their transcription rate can affect their folding. Here, we report the results of single-molecule fluorescence studies that characterize the ligand-dependent cotranscriptional folding of the Escherichia coli thiM riboswitch that regulates translation. We found that the riboswitch aptamer folds into the “off” conformation independent of its ligand, but switches to the “on” conformation during transcriptional pausing near the translational start codon. Ligand binding maintains the riboswitch in the off conformation during transcriptional pauses. We expect our assay will permit the controlled study of the two main physical mechanisms that regulate cotranscriptional folding: transcriptional pausing and transcriptional speed.

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

eLife ◽

10.7554/elife.44392 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 4

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.

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Quorum Sensing and Iron-Dependent Coordinated Control of Autoinducer-2 Production via Small RNA RyhB in Vibrio vulnificus

10.21203/rs.3.rs-936419/v1 ◽

2021 ◽

Author(s):

Keun-Woo Lee ◽

Yancheng Wen ◽

Na-Young Park ◽

Kun-Soo Kim

Keyword(s):

Quorum Sensing ◽

Small Rna ◽

Vibrio Vulnificus ◽

Iron Complex ◽

Start Codon ◽

Signal Pathways ◽

Dependent Manner ◽

Autoinducer 2 ◽

Translational Start Codon ◽

Translational Start

Abstract Roles for the non-coding small RNA RyhB in quorum-sensing and iron-dependent gene modulation in the human pathogen V. vulnificus were assessed in this study. Both the quorum sensing master regulator SmcR and the Fur-iron complex were observed to bind to the region upstream of the non-coding small RNA RyhB gene to repress expression, which suggests that RyhB is associated with both quorum-sensing and iron-dependent signaling in this pathogen. We found that expression of LuxS, which is responsible for the biosynthesis of autoinducer-2 (AI-2), was higher in wild type than in a ryhB-deletion isotype. RyhB binds directly to the 5'-UTR of the luxS transcript to form a heteroduplex, which not only stabilizes LuxS mRNA but also disrupts the secondary structure that normally obscures the translational start codon and thereby allows translation of LuxS to begin. The binding of RyhB to LuxS mRNA requires the chaperone protein Hfq, which stabilizes RyhB. These results demonstrate that the small RNA RyhB is a key element associated with feedback control of AI-2 production, and that it inhibits quorum-sensing signaling in an iron-dependent manner. This study, taken together with previous studies, shows that iron availability and cell density signals are funneled to SmcR and RyhB, and that these regulators coordinate cognate signal pathways that result in the proper balance of protein expression in response to environmental conditions.

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

10.1101/415018 ◽

2018 ◽

Cited By ~ 1

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.

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Characterization of Spodoptera exigua multicapsid nucleopolyhedrovirus ORF17/18, a homologue of Xestia c-nigrum granulovirus ORF129

Journal of General Virology ◽

10.1099/0022-1317-83-11-2857 ◽

2002 ◽

Vol 83 (11) ◽

pp. 2857-2867 ◽

Cited By ~ 1

Author(s):

Wilfred F. J. IJkel ◽

Els C. Roode ◽

Rob W. Goldbach ◽

Just M. Vlak ◽

Douwe Zuidema

Keyword(s):

Fusion Protein ◽

Spodoptera Exigua ◽

Polyclonal Antiserum ◽

Start Codon ◽

E Coli ◽

Gfp Fusion Protein ◽

Translational Start Codon ◽

Translational Start ◽

Race Analysis

Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) contains a number of genes with a homologue found so far only in a distantly related baculovirus. One of these, SeMNPV ORF17/18 (Se17/18) shares 55% amino acid similarity to ORF129 of Xestia c-nigrum granulovirus (XcGV). Se17/18 was transcribed in cultured S. exigua 301 cells, as a polyadenylated transcript of 1·1 kb. 5′-RACE analysis demonstrated that Se17/18 transcripts started at 134, 131 and 126 nt upstream of the putative translational start codon. These sites overlap with a baculovirus consensus early promoter motif. Se17/18 transcripts were detected by Northern blot analysis and RT–PCR with increasing abundance from 8 h to 24 h post infection (p.i.) and still present until 72 h p.i. A C-terminal GFP-fusion protein of Se17/18 was primarily localized in the cytoplasm of Se301 and Sf21 cells. A chicken polyclonal antiserum was raised that reacted specifically to Se17/18 protein produced in E. coli. However, no immunoreactive protein was detected in SeMNPV-infected Se301 cells and S. exigua larvae, neither in concentrated BV and ODV preparations. These observations and the inability to detect a C-terminal GFP-fusion protein of Se17/18 in Se301 cells using a GFP antibody suggest that Se17/18 protein is present, if at all, in spurious amounts. Based on the low homology of the Se17/18 protein to (methyl) transferases its possible involvement in transcription regulation is discussed.

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An A-Factor-Dependent Extracytoplasmic Function Sigma Factor (ςAdsA) That Is Essential for Morphological Development in Streptomyces griseus

Journal of Bacteriology ◽

10.1128/jb.182.16.4596-4605.2000 ◽

2000 ◽

Vol 182 (16) ◽

pp. 4596-4605 ◽

Cited By ~ 81

Author(s):

Haruka Yamazaki ◽

Yasuo Ohnishi ◽

Sueharu Horinouchi

Keyword(s):

Sigma Factor ◽

Streptomyces Griseus ◽

Transcriptional Activator ◽

Start Codon ◽

Morphological Development ◽

Aerial Hyphae ◽

Transcriptional Start Point ◽

Translational Start Codon ◽

Translational Start ◽

Nuclease Mapping

ABSTRACT A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) at an extremely low concentration triggers streptomycin production and aerial mycelium formation in Streptomyces griseus. A-factor induces the expression of an A-factor-dependent transcriptional activator, AdpA, essential for both morphological and physiological differentiation by binding to the A-factor receptor protein ArpA, which has bound and repressed the adpA promoter, and dissociating it from the promoter. Nine DNA fragments that were specifically recognized and bound by histidine-tagged AdpA were isolated by cycles of a gel mobility shift-PCR method. One of them was located in front of a gene encoding an extracytoplasmic function ς factor belonging to a subgroup of the primary ς70 family. The cloned gene was named AdpA-dependent sigma factor gene (adsA), and the gene product was named ςAdsA. Transcription ofadsA depended on A-factor and AdpA, since adsAwas transcribed at a very low and constant level in an A-factor-deficient mutant strain or in an adpA-disrupted strain. Consistent with this, transcription of adsA was greatly enhanced at or near the timing of aerial hyphae formation, as determined by low-resolution S1 nuclease mapping. High-resolution S1 mapping determined the transcriptional start point 82 nucleotides upstream of the translational start codon. DNase I footprinting showed that AdpA bound both strands symmetrically between the transcriptional start point and the translational start codon; AdpA protected the antisense strand from positions +7 to +41 with respect to the transcriptional start point and the sense strand from positions +12 to +46. A weak palindrome was found in the AdpA-binding site. The unusual position bound by AdpA as a transcriptional activator, in relation to the promoter, suggested the presence of a mechanism by which AdpA activates transcription of adsA in some unknown way. Disruption of the chromosomal adsA gene resulted in loss of aerial hyphae formation but not streptomycin or yellow pigment production, indicating that ςAdsA is involved only in morphological development and not in secondary metabolic function. The presence of a single copy in each of the Streptomycesspecies examined by Southern hybridization suggests a common role in morphogenesis in this genus.

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

10.1101/031625 ◽

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.

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

10.1101/852251 ◽

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.

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