A Coding Sequence-Embedded Principle Governs Translational Reading Frame Fidelity

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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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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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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Noncoding AUG circRNAs constitute an abundant and conserved subclass of circles

Life Science Alliance ◽

10.26508/lsa.201900398 ◽

2019 ◽

Vol 2 (3) ◽

pp. e201900398 ◽

Cited By ~ 23

Author(s):

Lotte VW Stagsted ◽

Katrine M Nielsen ◽

Iben Daugaard ◽

Thomas B Hansen

Keyword(s):

Ribosome Profiling ◽

Start Codon ◽

Circular Rnas ◽

Protein Coding ◽

Reading Frame ◽

Flanking Sequences ◽

Bona Fide ◽

Translational Start Codon ◽

Comprehensive Classification ◽

Translational Start

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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Temperature-Dependent Regulation of Upstream Open Reading Frame Translation inS. Cerevisiae

10.1101/678409 ◽

2019 ◽

Cited By ~ 1

Author(s):

Shardul D. Kulkarni ◽

Fujun Zhou ◽

Neelam Dabas Sen ◽

Hongen Zhang ◽

Alan G. Hinnebusch ◽

...

Keyword(s):

Gene Expression ◽

Growth Temperature ◽

Ribosome Profiling ◽

Open Reading Frames ◽

Yeast Cells ◽

Upstream Open Reading Frame ◽

Start Codon ◽

Translational Efficiency ◽

Temperature Dependent ◽

Reading Frame

AbstractBackgroundTranslation of an mRNA in eukaryotes starts at AUG in most cases. Near-cognate codons (NCCs) such as UUG, ACG and AUU are also used as start sites at low levels inS. cerevisiae. Initiation from NCCs or AUGs in the 5’-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature.ResultsUsing reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs inS. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 °C relative to 30 °C and decreased efficiency at 20 °C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5’-leader relative to the 5’-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation.ConclusionsOverall, our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression inS. cerevisiae.

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Translation of 5′ leaders is pervasive in genes resistant to eIF2 repression

eLife ◽

10.7554/elife.03971 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 171

Author(s):

Dmitry E Andreev ◽

Patrick BF O'Connor ◽

Ciara Fahey ◽

Elaine M Kenny ◽

Ilya M Terenin ◽

...

Keyword(s):

Ribosome Profiling ◽

Open Reading Frames ◽

Translation Initiation Factor ◽

Upstream Open Reading Frame ◽

Initiation Factor ◽

Translation Control ◽

Functional Protein ◽

Protein Coding ◽

Reading Frame ◽

Initiation Factor 2

Eukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated upstream open reading frame (uORF) that represses translation of the main coding ORF under normal conditions. Site-specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products.

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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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Persistence of ambigrammatic narnaviruses requires translation of the reverse open reading frame

Journal of Virology ◽

10.1128/jvi.00109-21 ◽

2021 ◽

Author(s):

Hanna Retallack ◽

Katerina D. Popova ◽

Matthew T. Laurie ◽

Sara Sunshine ◽

Joseph L. DeRisi

Keyword(s):

Viral Genome ◽

Rna Viruses ◽

Single Gene ◽

Ribosome Profiling ◽

Open Reading Frames ◽

Reading Frame ◽

Infected Cells ◽

The Cost ◽

Overlapping Open Reading Frames ◽

First Time

Narnaviruses are RNA viruses detected in diverse fungi, plants, protists, arthropods and nematodes. Though initially described as simple single-gene non-segmented viruses encoding RNA-dependent RNA polymerase (RdRp), a subset of narnaviruses referred to as “ambigrammatic” harbor a unique genomic configuration consisting of overlapping open reading frames (ORFs) encoded on opposite strands. Phylogenetic analysis supports selection to maintain this unusual genome organization, but functional investigations are lacking. Here, we establish the mosquito-infecting Culex narnavirus 1 (CxNV1) as a model to investigate the functional role of overlapping ORFs in narnavirus replication. In CxNV1, a reverse ORF without homology to known proteins covers nearly the entire 3.2 kb segment encoding the RdRp. Additionally, two opposing and nearly completely overlapping novel ORFs are found on the second putative CxNV1 segment, the 0.8 kb “Robin” RNA. We developed a system to launch CxNV1 in a naïve mosquito cell line, then showed that functional RdRp is required for persistence of both segments, and an intact reverse ORF is required on the RdRp segment for persistence. Mass spectrometry of persistently CxNV1-infected cells provided evidence for translation of this reverse ORF. Finally, ribosome profiling yielded a striking pattern of footprints for all four CxNV1 RNA strands that was distinct from actively-translating ribosomes on host mRNA or co-infecting RNA viruses. Taken together, these data raise the possibility that the process of translation itself is important for persistence of ambigrammatic narnaviruses, potentially by protecting viral RNA with ribosomes, thus suggesting a heretofore undescribed viral tactic for replication and transmission. IMPORTANCE Fundamental to our understanding of RNA viruses is a description of which strand(s) of RNA are transmitted as the viral genome, relative to which encode the viral proteins. Ambigrammatic narnaviruses break the mold. These viruses, found broadly in fungi, plants, and insects, have the unique feature of two overlapping genes encoded on opposite strands, comprising nearly the full length of the viral genome. Such extensive overlap is not seen in other RNA viruses, and comes at the cost of reduced evolutionary flexibility in the sequence. The present study is motivated by investigating the benefits which balance that cost. We show for the first time a functional requirement for the ambigrammatic genome configuration in Culex narnavirus 1, which suggests a model for how translation of both strands might benefit this virus. Our work highlights a new blueprint for viral persistence, distinct from strategies defined by canonical definitions of the coding strand.

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FuncPEP: A Database of Functional Peptides Encoded by Non-Coding RNAs

Non-Coding RNA ◽

10.3390/ncrna6040041 ◽

2020 ◽

Vol 6 (4) ◽

pp. 41

Author(s):

Mihnea P. Dragomir ◽

Ganiraju C. Manyam ◽

Leonie Florence Ott ◽

Léa Berland ◽

Erik Knutsen ◽

...

Keyword(s):

Open Reading Frames ◽

Start Codon ◽

Small Peptides ◽

Reading Frame ◽

Cellular Processes ◽

New Class ◽

Web Resource ◽

Non Coding Rnas ◽

Functional Peptides ◽

Small Open Reading Frames

Non-coding RNAs (ncRNAs) are essential players in many cellular processes, from normal development to oncogenic transformation. Initially, ncRNAs were defined as transcripts that lacked an open reading frame (ORF). However, multiple lines of evidence suggest that certain ncRNAs encode small peptides of less than 100 amino acids. The sequences encoding these peptides are known as small open reading frames (smORFs), many initiating with the traditional AUG start codon but terminating with atypical stop codons, suggesting a different biogenesis. The ncRNA-encoded peptides (ncPEPs) are gradually becoming appreciated as a new class of functional molecules that contribute to diverse cellular processes, and are deregulated in different diseases contributing to pathogenesis. As multiple publications have identified unique ncPEPs, we appreciated the need for assembling a new web resource that could gather information about these functional ncPEPs. We developed FuncPEP, a new database of functional ncRNA encoded peptides, containing all experimentally validated and functionally characterized ncPEPs. Currently, FuncPEP includes a comprehensive annotation of 112 functional ncPEPs and specific details regarding the ncRNA transcripts that encode these peptides. We believe that FuncPEP will serve as a platform for further deciphering the biologic significance and medical use of ncPEPs. The link for FuncPEP database can be found at the end of the Introduction Section.

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Recommendations for bacterial ribosome profiling experiments based on bioinformatic evaluation of published data

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012161 ◽

2020 ◽

Vol 295 (27) ◽

pp. 8999-9011 ◽

Cited By ~ 2

Author(s):

Alina Glaub ◽

Christopher Huptas ◽

Klaus Neuhaus ◽

Zachary Ardern

Keyword(s):

Ribosome Profiling ◽

Published Data ◽

Data Sets ◽

Drug Induced ◽

Data Set ◽

Protein Coding ◽

Bacterial Ribosome ◽

Translation Start ◽

Selection Step ◽

Basic Characteristics

Ribosome profiling (RIBO-Seq) has improved our understanding of bacterial translation, including finding many unannotated genes. However, protocols for RIBO-Seq and corresponding data analysis are not yet standardized. Here, we analyzed 48 RIBO-Seq samples from nine studies of Escherichia coli K12 grown in lysogeny broth medium and particularly focused on the size-selection step. We show that for conventional expression analysis, a size range between 22 and 30 nucleotides is sufficient to obtain protein-coding fragments, which has the advantage of removing many unwanted rRNA and tRNA reads. More specific analyses may require longer reads and a corresponding improvement in rRNA/tRNA depletion. There is no consensus about the appropriate sequencing depth for RIBO-Seq experiments in prokaryotes, and studies vary significantly in total read number. Our analysis suggests that 20 million reads that are not mapping to rRNA/tRNA are required for global detection of translated annotated genes. We also highlight the influence of drug-induced ribosome stalling, which causes bias at translation start sites. The resulting accumulation of reads at the start site may be especially useful for detecting weakly expressed genes. As different methods suit different questions, it may not be possible to produce a “one-size-fits-all” ribosome profiling data set. Therefore, experiments should be carefully designed in light of the scientific questions of interest. We propose some basic characteristics that should be reported with any new RIBO-Seq data sets. Careful attention to the factors discussed should improve prokaryotic gene detection and the comparability of ribosome profiling data sets.

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Genome-wide identification of Arabidopsis non-AUG-initiated upstream ORFs with evolutionarily conserved regulatory sequences that control protein expression levels

10.1101/2021.03.25.436978 ◽

2021 ◽

Author(s):

Yuta Hiragori ◽

Hiro Takahashi ◽

Noriya Hayashi ◽

Shun Sasaki ◽

Kodai Nakao ◽

...

Keyword(s):

Regulatory Peptides ◽

Inhibitory Effect ◽

Ribosome Profiling ◽

Open Reading Frames ◽

Regulatory Sequences ◽

Dependent Manner ◽

Protein Coding ◽

Genome Wide ◽

Evolutionarily Conserved ◽

Upstream Open Reading Frames

Upstream open reading frames (uORFs) are short ORFs found in the 5′-UTRs of many eukaryotic transcripts and can influence the translation of protein-coding main ORFs (mORFs). Recent genome-wide ribosome profiling studies have revealed that thousands of uORFs initiate translation at non-AUG start codons. However, the physiological significance of these non-AUG uORFs has so far been demonstrated for only a few of them. It is conceivable that physiologically important non-AUG uORFs are evolutionarily conserved across species. In this study, using a combination of bioinformatics and experimental approaches, we searched the Arabidopsis genome for non-AUG-initiated uORFs with conserved sequences that control the expression of the mORF-encoded proteins. As a result, we identified four novel regulatory non-AUG uORFs. Among these, two exerted repressive effects on mORF expression in an amino acid sequence-dependent manner. These two non-AUG uORFs are likely to encode regulatory peptides that cause ribosome stalling, thereby enhancing their repressive effects. In contrast, one of the identified regulatory non-AUG uORFs promoted mORF expression by alleviating the inhibitory effect of a downstream AUG-initiated uORF. These findings provide insights into the mechanisms that enable non-AUG uORFs to play regulatory roles despite their low translation initiation efficiencies.

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