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 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.

The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences

1988 ◽  
Vol 8 (9) ◽  
pp. 3827-3836
Author(s):  
N P Williams ◽  
P P Mueller ◽  
A G Hinnebusch
Keyword(s):  
Translational Control ◽  
Normal Growth ◽  
Regulatory Elements ◽  
Growth Conditions ◽  
Open Reading Frames ◽  
Regulatory Function ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
Yeast Genes ◽  
Reading Frames

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.

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.

The first and fourth upstream open reading frames in GCN4 mRNA have similar initiation efficiencies but respond differently in translational control to change in length and sequence

1988 ◽  
Vol 8 (12) ◽  
pp. 5439-5447
Author(s):  
P P Mueller ◽  
B M Jackson ◽  
P F Miller ◽  
A G Hinnebusch
Keyword(s):  
Translational Control ◽  
Normal Growth ◽  
Open Reading Frames ◽  
Regulatory Function ◽  
Coding Sequences ◽  
Lacz Fusions ◽  
Upstream Open Reading Frames ◽  
Reading Frames ◽  
Starvation Conditions

The third and fourth AUG codons in GCN4 mRNA efficiently repress translation of the GCN4-coding sequences under normal growth conditions. The first AUG codon is approximately 30-fold less inhibitory and is required under amino acid starvation conditions to override the repressing effects of AUG codons 3 and 4. lacZ fusions constructed to functional, elongated versions of the first and fourth upstream open reading frames (URFs) were used to show that AUG codons 1 and 4 function similarly as efficient translational start sites in vivo, raising the possibility that steps following initiation distinguish the regulatory properties of URFs 1 and 4. In accord with this idea, we observed different consequences of changing the length and termination site of URF1 versus changing those of URFs 3 and 4. The latter were lengthened considerably, with little or no effect on regulation. In fact, the function of URFs 3 and 4 was partially reconstituted with a completely heterologous URF. By contrast, certain mutations that lengthen URF1 impaired its positive regulatory function nearly as much as removing its AUG codon did. The same mutations also made URF1 a much more inhibitory element when it was present alone in the mRNA leader. These results strongly suggest that URFs 1 and 4 both function in regulation as translated coding sequences. To account for the phenotypes of the URF1 mutations, we suggest the most ribosomes normally translate URF1 and that the mutations reduce the number of ribosomes that are able to complete URF1 translation and resume scanning downstream. This effect would impair URF1 positive regulatory function if ribosomes must first translate URF1 in order to overcome the strong translational block at the 3'-proximal URFs. Because URF1-lacZ fusions were translated at the same rate under repressing and derepressing conditions, it appears that modulating initiation at URF1 is not the means that is used to restrict the regulatory consequences of URF1 translation to starvation conditions.

scholarly journals Inhibition of translation of transforming growth factor-beta 3 mRNA by its 5' untranslated region.

1991 ◽  
Vol 11 (9) ◽  
pp. 4306-4313 ◽  
Author(s):  
B A Arrick ◽  
A L Lee ◽  
R L Grendell ◽  
R Derynck
Keyword(s):  
Transforming Growth Factor Beta ◽  
Untranslated Region ◽  
Transforming Growth Factor ◽  
Open Reading Frames ◽  
In Vitro Translation ◽  
Tgf Beta ◽  
Coding Sequence ◽  
Upstream Open Reading Frames ◽  
Reading Frames

We have cloned and sequenced the 5' untranslated region of the transforming growth factor-beta 3 (TGF-beta 3) mRNA as well as the adjacent genomic sequence. S1 nuclease analysis identified a single transcription start site. We have thus determined that the 5' untranslated region is about 1.1 kb long and contains 11 open reading frames. In vitro translation of the TGF-beta 3 precursor coding sequence was markedly inhibited by the presence of the 5' untranslated region. Similarly, when the 5' untranslated region of TGF-beta 3 was introduced upstream of the coding sequence of chloramphenicol acetyltransferase, in vitro translation was inhibited. Furthermore, upon transfection into 293 cells, chloramphenicol acetyltransferase expression was inhibited by the 5' untranslated region of TGF-beta 3. The degree of translational inhibition was inversely proportional to the amount of transfected DNA. Mutation analysis implicated multiple segments of the 5' untranslated region as contributing to the inhibitory effect. Deletion of much of the 5'-most 640 nucleotides, including 8 of the 11 upstream ATGs, relieved much but not all of the inhibitory influence of the 5' untranslated region of TGF-beta 3 mRNA. The two upstream open reading frames closest to the initiator codon for the TGF-beta 3 coding sequence also decreased translational efficiency, since mutation of either ATG resulted in increased translation. Transfection results with T47-D cells, a cell line which expresses TGF-beta 3 mRNA, were similar to those obtained with the 293 cell line. Thus, TGF-beta 3 mRNA is a recent example of an expanding group of growth-related mRNAs in which the 5' untranslated region contains upstream open reading frames and other sequences which inhibit translation.

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 Translational Control of Protein Kinase Cη by Two Upstream Open Reading Frames

2009 ◽  
Vol 29 (22) ◽  
pp. 6140-6148 ◽  
Author(s):  
Hadas Raveh-Amit ◽  
Adva Maissel ◽  
Jonathan Poller ◽  
Liraz Marom ◽  
Orna Elroy-Stein ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Kinase ◽  
Translational Control ◽  
Translational Regulation ◽  
Normal Growth ◽  
Amino Acid Starvation ◽  
Open Reading Frames ◽  
Pkc Isoforms ◽  
Upstream Open Reading Frames ◽  
Reading Frames

ABSTRACT Protein kinase C (PKC) represents a family of serine/threonine kinases that play a central role in the regulation of cell growth, differentiation, and transformation. Posttranslational control of the PKC isoforms and their activation have been extensively studied; however, not much is known about their translational regulation. Here we report that the expression of one of the PKC isoforms, PKCη, is regulated at the translational level both under normal growth conditions and during stress imposed by amino acid starvation, the latter causing a marked increase in its protein levels. The 5′ untranslated region (5′ UTR) of PKCη is unusually long and GC rich, characteristic of many oncogenes and growth regulatory genes. We have identified two conserved upstream open reading frames (uORFs) in its 5′ UTR and show their effect in suppressing the expression of PKCη in MCF-7 growing cells. While the two uORFs function as repressive elements that maintain low basal levels of PKCη in growing cells, they are required for its enhanced expression upon amino acid starvation. We show that the translational regulation during stress involves leaky scanning and is dependent on eIF-2α phosphorylation by GCN2. Our work further suggests that translational regulation could provide an additional level for controlling the expression of PKC family members, being more common than currently recognized.

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