scholarly journals Systematic analysis of the PTEN 5′ leader identifies a major AUU initiated proteoform

Open Biology ◽  
2016 ◽  
Vol 6 (5) ◽  
pp. 150203 ◽  
Author(s):  
Ioanna Tzani ◽  
Ivaylo P. Ivanov ◽  
Dmitri E. Andreev ◽  
Ruslan I. Dmitriev ◽  
Kellie A. Dean ◽  
...  
Keyword(s):  
Pi3k Pathway ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Sequencing Data ◽  
Human Tumour ◽  
Systematic Analysis ◽  
Human Genes ◽  
Abundant Evidence ◽  
Upstream Open Reading Frames ◽  
Reading Frames

Abundant evidence for translation within the 5′ leaders of many human genes is rapidly emerging, especially, because of the advent of ribosome profiling. In most cases, it is believed that the act of translation rather than the encoded peptide is important. However, the wealth of available sequencing data in recent years allows phylogenetic detection of sequences within 5′ leaders that have emerged under coding constraint and therefore allow for the prediction of functional 5′ leader translation. Using this approach, we previously predicted a CUG-initiated, 173 amino acid N-terminal extension to the human tumour suppressor PTEN. Here, a systematic experimental analysis of translation events in the PTEN 5′ leader identifies at least two additional non-AUG-initiated PTEN proteoforms that are expressed in most human cell lines tested. The most abundant extended PTEN proteoform initiates at a conserved AUU codon and extends the canonical AUG-initiated PTEN by 146 amino acids. All N-terminally extended PTEN proteoforms tested retain the ability to downregulate the PI3K pathway. We also provide evidence for the translation of two conserved AUG-initiated upstream open reading frames within the PTEN 5′ leader that control the ratio of PTEN proteoforms.

scholarly journals Translation of ABCE1 Is Tightly Regulated by Upstream Open Reading Frames in Human Colorectal Cells

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 911
Author(s):  
Joana Silva ◽  
Pedro Nina ◽  
Luísa Romão
Keyword(s):  
Translational Regulation ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Leader Sequence ◽  
Open Reading Frames ◽  
Regulatory Function ◽  
Coding Sequence ◽  
Abc Protein ◽  
Upstream Open Reading Frames ◽  
Reading Frames

ATP-binding cassette subfamily E member 1 (ABCE1) belongs to the ABC protein family of transporters; however, it does not behave as a drug transporter. Instead, ABCE1 actively participates in different stages of translation and is also associated with oncogenic functions. Ribosome profiling analysis in colorectal cancer cells has revealed a high ribosome occupancy in the human ABCE1 mRNA 5′-leader sequence, indicating the presence of translatable upstream open reading frames (uORFs). These cis-acting translational regulatory elements usually act as repressors of translation of the main coding sequence. In the present study, we dissect the regulatory function of the five AUG and five non-AUG uORFs identified in the human ABCE1 mRNA 5′-leader sequence. We show that the expression of the main coding sequence is tightly regulated by the ABCE1 AUG uORFs in colorectal cells. Our results are consistent with a model wherein uORF1 is efficiently translated, behaving as a barrier to downstream uORF translation. The few ribosomes that can bypass uORF1 (and/or uORF2) must probably initiate at the inhibitory uORF3 or uORF5 that efficiently repress translation of the main ORF. This inhibitory property is slightly overcome in conditions of endoplasmic reticulum stress. In addition, we observed that these potent translation-inhibitory AUG uORFs function equally in cancer and in non-tumorigenic colorectal cells, which is consistent with a lack of oncogenic function. In conclusion, we establish human ABCE1 as an additional example of uORF-mediated translational regulation and that this tight regulation contributes to control ABCE1 protein levels in different cell environments.

scholarly journals Disrupting upstream translation in mRNAs is associated with human disease

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.

scholarly journals Improved ribosome-footprint and mRNA measurements provide insights into dynamics and regulation of yeast translation

2015 ◽  
Author(s):  
David E Weinberg ◽  
Premal Shah ◽  
Stephen W Eichhorn ◽  
Jeffrey A Hussmann ◽  
Joshua B Plotkin ◽  
...  
Keyword(s):  
Translational Control ◽  
Nucleotide Composition ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Untranslated Regions ◽  
Coding Regions ◽  
Genome Wide ◽  
Upstream Open Reading Frames ◽  
Improved Methods ◽  
Reading Frames

Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a 10-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5′- untranslated regions. Collectively, our results reveal key features of translational control in yeast and provide a framework for executing and interpreting ribosome- profiling studies.

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 Autonomous functionality of an upstream open reading frame in polycistronic mammalian mRNAs

2018 ◽  
Author(s):  
Shohei Kitano ◽  
Gabriel Pratt ◽  
Keizo Takao ◽  
Yasunori Aizawa
Keyword(s):  
Brain Regions ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Upstream Open Reading Frame ◽  
Translation Regulation ◽  
Reading Frame ◽  
Eukaryotic Translation ◽  
Upstream Open Reading Frames ◽  
Human And Mouse ◽  
Reading Frames

SUMMARYUpstream open reading frames (uORFs) are established as cis-acting elements for eukaryotic translation of annotated ORFs (anORFs) located on the same mRNAs. Here, we identified a mammalian uORF with functions that are independent from anORF translation regulation. Bioinformatics screening using ribosome profiling data of human and mouse brains yielded 308 neurologically vital genes from which anORF and uORFs are polycistronically translated in both species. Among them, Arhgef9 contains a uORF named SPICA, which is highly conserved among vertebrates and stably translated only in specific brain regions of mice. Disruption of SPICA translation by ATG-to-TAG substitutions did not perturb translation or function of its anORF product, collybistin. SPICA-null mice displayed abnormal maternal reproductive performance and enhanced anxiety-like behavior, characteristic of ARHGEF9-associated neurological disorders. This study demonstrates that mammalian uORFs can be independent genetic units, revising the prevailing dogma of the monocistronic gene in mammals, and even eukaryotes.

scholarly journals RNA G-quadruplexes mark repressive upstream open reading frames in human mRNAs

2017 ◽  
Author(s):  
Pierre Murat ◽  
Giovanni Marsico ◽  
Barbara Herdy ◽  
Avazeh Ghanbarian ◽  
Guillem Portella ◽  
...  
Keyword(s):  
Secondary Structures ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Untranslated Regions ◽  
Translation Regulation ◽  
Physical Interaction ◽  
Protein Coding ◽  
Upstream Open Reading Frames ◽  
Nucleotide Resolution ◽  
Reading Frames

ABSTRACTRNA secondary structures in the 5’ untranslated regions (UTRs) of mRNAs have been characterised as key determinants of translation initiation. However the role of non-canonical secondary structures, such as RNA G-quadruplexes (rG4s), in modulating translation of human mRNAs and the associated mechanisms remain largely unappreciated. Here we use a ribosome profiling strategy to investigate the translational landscape of human mRNAs with structured 5’ untranslated regions (5’-UTR). We found that inefficiently translated mRNAs, containing rG4-forming sequences in their 5’-UTRs, have an accumulation of ribosome footprints in their 5’-UTRs. We show that rG4-forming sequences are determinants of 5’-UTR translation, suggesting that the folding of rG4 structures thwarts the translation of protein coding sequences (CDS) by stimulating the translation of repressive upstream open reading frames (uORFs). To support our model, we demonstrate that depletion of two rG4s-specialised DEAH-box helicases, DHX36 and DHX9, shifts translation towards rG4-containing uORFs reducing the translation of selected transcripts comprising proto-oncogenes, transcription factors and epigenetic regulators. Transcriptome-wide identification of DHX9 binding sites using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) demonstrate that translation regulation is mediated through direct physical interaction between the helicase and its rG4 substrate. Our findings unveil a previously unknown role for non-canonical structures in governing 5’-UTR translation and suggest that the interaction of helicases with rG4s could be considered as a target for future therapeutic intervention.

scholarly journals Ribosome profiling reveals the rhythmic liver translatome and circadian clock regulation by upstream open reading frames

2015 ◽  
Vol 25 (12) ◽  
pp. 1848-1859 ◽  
Author(s):  
Peggy Janich ◽  
Alaaddin Bulak Arpat ◽  
Violeta Castelo-Szekely ◽  
Maykel Lopes ◽  
David Gatfield
Keyword(s):  
Circadian Clock ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Upstream Open Reading Frames ◽  
Reading Frames

scholarly journals Upstream Open Reading Frames (uORFs) in the Apicomplexan Parasites Plasmodium falciparum and Toxoplasma gondii: Small yet Powerful Regulators of Translation

2021 ◽  
Author(s):  
Chhaminder Kaur ◽  
Swati Patankar
Keyword(s):  
Gene Expression ◽  
Plasmodium Falciparum ◽  
Toxoplasma Gondii ◽  
Translational Regulation ◽  
Life Cycles ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Apicomplexan Parasites ◽  
Upstream Open Reading Frames ◽  
Reading Frames

During their complex life cycles, the Apicomplexan parasites, Plasmodium falciparum and Toxoplasma gondii employ several genetic switches to regulate their gene expression. One such switch is mediated at the level of translation through upstream Open Reading Frames (uORFs). As uORFs are found in the upstream regions of a majority of genes in both the parasites, it is essential that their roles in translational regulation be appreciated to a greater extent. This review provides a comprehensive summary of studies that show uORF-mediated gene regulation in these parasites and highlights examples of clinically and physiologically relevant proteins that exhibit uORF-mediated regulation. In addition to these examples, several studies that use bioinformatics, transcriptomics, proteomics, and ribosome profiling also indicate the possibility of widespread translational regulation by uORFs. Further analysis of genome-wide datasets will reveal novel genes involved in key biological pathways such as cell-cycle progression, stress-response, and pathogenicity. The cumulative evidence from studies presented in this review suggests that uORFs will play crucial roles in regulating gene expression during clinical disease caused by these important human pathogens.

scholarly journals ORFik: a comprehensive R toolkit for the analysis of translation

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Håkon Tjeldnes ◽  
Kornel Labun ◽  
Yamila Torres Cleuren ◽  
Katarzyna Chyżyńska ◽  
Michał Świrski ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Sequencing Data ◽  
Protein Coding ◽  
High Throughput Sequencing Data ◽  
Upstream Open Reading Frames ◽  
Many Core ◽  
User Friendly

Abstract Background With the rapid growth in the use of high-throughput methods for characterizing translation and the continued expansion of multi-omics, there is a need for back-end functions and streamlined tools for processing, analyzing, and characterizing data produced by these assays. Results Here, we introduce ORFik, a user-friendly R/Bioconductor API and toolbox for studying translation and its regulation. It extends GenomicRanges from the genome to the transcriptome and implements a framework that integrates data from several sources. ORFik streamlines the steps to process, analyze, and visualize the different steps of translation with a particular focus on initiation and elongation. It accepts high-throughput sequencing data from ribosome profiling to quantify ribosome elongation or RCP-seq/TCP-seq to also quantify ribosome scanning. In addition, ORFik can use CAGE data to accurately determine 5′UTRs and RNA-seq for determining translation relative to RNA abundance. ORFik supports and calculates over 30 different translation-related features and metrics from the literature and can annotate translated regions such as proteins or upstream open reading frames (uORFs). As a use-case, we demonstrate using ORFik to rapidly annotate the dynamics of 5′ UTRs across different tissues, detect their uORFs, and characterize their scanning and translation in the downstream protein-coding regions. Conclusion In summary, ORFik introduces hundreds of tested, documented and optimized methods. ORFik is designed to be easily customizable, enabling users to create complete workflows from raw data to publication-ready figures for several types of sequencing data. Finally, by improving speed and scope of many core Bioconductor functions, ORFik offers enhancement benefiting the entire Bioconductor environment. Availability http://bioconductor.org/packages/ORFik.

scholarly journals ORFik: a comprehensive R toolkit for the analysis of translation

2021 ◽  
Author(s):  
Håkon Tjeldnes ◽  
Kornel Labun ◽  
Yamila Torres Cleuren ◽  
Katarzyna Chyżyńska ◽  
Michał Świrski ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Sequencing Data ◽  
Protein Coding ◽  
Coding Regions ◽  
High Throughput Sequencing Data ◽  
Upstream Open Reading Frames ◽  
User Friendly

ABSTRACT•BackgroundWith the rapid growth in the use of high-throughput methods for characterizing translation and the continued expansion of multi-omics, there is a need for back-end functions and streamlined tools for processing, analyzing, and characterizing data produced by these assays.•ResultsHere, we introduce ORFik, a user-friendly R/Bioconductor toolbox for studying translation and its regulation. It extends GenomicRanges from the genome to the transcriptome and implements a framework that integrates data from several sources. ORFik streamlines the steps to process, analyze, and visualize the different steps of translation with a particular focus on initiation and elongation. It accepts high-throughput sequencing data from ribosome profiling to quantify ribosome elongation or RCP-seq/TCP-seq to also quantify ribosome scanning. In addition, ORFik can use CAGE data to accurately determine 5’UTRs and RNA-seq for determining translation relative to RNA abundance. ORFik supports and calculates over 30 different translation-related features and metrics from the literature and can annotate translated regions such as proteins or upstream open reading frames. As a use-case, we demonstrate using ORFik to rapidly annotate the dynamics of 5’ UTRs across different tissues, detect their uORFs, and characterize their scanning and translation in the downstream protein-coding regions.•Availabilityhttp://bioconductor.org/packages/ORFik

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