Ribosome profiling reveals the rhythmic liver translatome and circadian clock regulation by upstream open 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.

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

10.1101/021501 ◽

2015 ◽

Cited By ~ 2

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.

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

10.1101/325571 ◽

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.

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

10.1101/223073 ◽

2017 ◽

Cited By ~ 1

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.

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

10.20944/preprints202104.0680.v1 ◽

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.

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

Open Biology ◽

10.1098/rsob.150203 ◽

2016 ◽

Vol 6 (5) ◽

pp. 150203 ◽

Cited By ~ 20

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.

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uORF Shuffling Fine-Tunes Gene Expression at a Deep Level of the Process

Plants ◽

10.3390/plants9050608 ◽

2020 ◽

Vol 9 (5) ◽

pp. 608

Author(s):

Yukio Kurihara

Keyword(s):

Gene Expression ◽

Deep Level ◽

Ribosome Profiling ◽

Open Reading Frames ◽

Protein Coding ◽

Polycistronic Mrna ◽

Upstream Open Reading Frames ◽

Fine Tune ◽

Insight Into ◽

Reading Frames

Upstream open reading frames (uORFs) are present in the 5’ leader sequences (or 5’ untranslated regions) upstream of the protein-coding main ORFs (mORFs) in eukaryotic polycistronic mRNA. It is well known that a uORF negatively affects translation of the mORF. Emerging ribosome profiling approaches have revealed that uORFs themselves, as well as downstream mORFs, can be translated. However, it has also been revealed that plants can fine-tune gene expression by modulating uORF-mediated regulation in some situations. This article reviews several proposed mechanisms that enable genes to escape from uORF-mediated negative regulation and gives insight into the application of uORF-mediated regulation for precisely controlling gene expression.

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Translation of upstream open reading frames in a model of neuronal differentiation

10.1101/412106 ◽

2018 ◽

Cited By ~ 1

Author(s):

C.M. Rodriguez ◽

S.Y. Chun ◽

R.E. Mills ◽

P.K. Tod

Keyword(s):

Neuronal Differentiation ◽

Neuroblastoma Cells ◽

Ribosome Profiling ◽

Open Reading Frames ◽

Model Systems ◽

Human Neuroblastoma ◽

Upstream Open Reading Frames ◽

Dynamic Relationships ◽

Regulatory Functions ◽

Reading Frames

AbstractUpstream open reading frames (uORFs) initiate translation within mRNA 5’ leaders, and have the potential to alter main coding sequence (CDS) translation on transcripts in which they reside. Ribosome profiling (RP) studies suggest that translating ribosomes are pervasive within 5’ leaders across model systems. However, the significance of this observation remains unclear. To explore a role for uORF usage in neuronal differentiation, we performed RP on undifferentiated and differentiated human neuroblastoma cells. Using a spectral coherence algorithm (SPECtre), we identify 4,954 uORFs across 31% of all neuroblastoma transcripts. These uORFs predominantly utilize non-AUG initiation codons and exhibit translational efficiencies (TE) comparable to annotated coding regions. Usage of both AUG initiated uORFs and a conserved and consistently translated subset of non-AUG initiated uORFs correlates with repressed CDS translation. Ribosomal protein transcripts are enriched in uORFs, and select uORFs on such transcripts were validated for expression. With neuronal differentiation, we observed an overall positive correlation between translational shifts in uORF/CDS pairs. However, a subset of transcripts exhibit inverse shifts in translation of uORF/CDS pairs. These uORFs are enriched in AUG initiation sites, non-overlapping, and shorter in length.Cumulatively, CDSs downstream of uORFs characterized by persistent translation show smaller shifts in TE with neuronal differentiation relative to CDSs without a predicted uORF, suggesting that fluctuations in CDS translation are buffered by uORF translation. In sum, this work provides insights into the dynamic relationships and potential regulatory functions of uORF/CDS pairs in a model of neuronal differentiation.

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DAP5 enables translation re-initiation on structured messenger RNAs

10.1101/2021.01.21.427569 ◽

2021 ◽

Author(s):

Ramona Weber ◽

Leon Kleemann ◽

Insa Hirschberg ◽

Min-Yi Chung ◽

Eugene Valkov ◽

...

Keyword(s):

Cell Fate ◽

Molecular Mechanisms ◽

Mutational Analysis ◽

Ribosome Profiling ◽

Open Reading Frames ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Messenger Rnas ◽

Upstream Open Reading Frames ◽

Reading Frames

SummaryHalf of mammalian transcripts contain short upstream open reading frames (uORFs) that potentially regulate translation of the downstream coding sequence (CDS). The molecular mechanisms governing these events remain poorly understood. Here we find that the non-canonical initiation factor Death-associated protein 5 (DAP5 or eIF4G2) is selectively required for re-initiation at the main CDS following uORF translation. Using ribosome profiling and luciferase-based reporters coupled with mutational analysis we show that DAP5-mediated re-initiation occurs on messenger RNAs (mRNAs) with long, structure-prone 5′ leader sequences and persistent uORF translation. These mRNAs preferentially code for signalling factors such as kinases and phosphatases. We also report that cap/eIF4F- and eIF4A-dependent recruitment of DAP5 to the mRNA facilitates re-initiation by unrecycled post-termination 40S subunits. Our study reveals important mechanistic insights into how a non-canonical translation initiation factor involved in stem cell fate shapes the synthesis of specific signalling factors.

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