scholarly journals Ribosomal profiling as a tool for studying translation in plants: main results, problems and future prospects

2021 ◽  
Vol 25 (3) ◽  
pp. 251-259
Author(s):  
D. A. Afonnikov ◽  
O. I. Sinitsyna ◽  
T. S. Golubeva ◽  
N. A. Shmakov ◽  
A. V. Kochetov
Keyword(s):  
High Performance ◽  
Functional Organization ◽  
Gc Content ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Translation Efficiency ◽  
Mrna Structure ◽  
Eukaryotic Genes ◽  
Plant Genes ◽  
Reading Frames

The expression of eukaryotic genes can be regulated at several stages, including the translation of mRNA. It is known that the structure of mRNA can affect both the efficiency of interaction with the translation apparatus in general and the choice of translation initiation sites. To study the translated fraction of the transcriptome, experimental methods of analysis were developed, the most informative of which is ribosomal profiling (RP, Ribo-seq). Originally developed for use in yeast systems, this method has been adapted for research in translation mechanisms in many plant species. This technology includes the isolation of the polysomal fraction and high-performance sequencing of a pool of mRNA fragments associated with ribosomes. Comparing the results of transcript coverage with reads obtained using the ribosome profiling with the transcriptional efficiency of genes allows the translation efficiency to be evaluated for each transcript. The exact positions of ribosomes determined on mRNA sequences allow determining the translation of open reading frames and switching between the translation of several reading frames – a phenomenon in which two or more overlapping frames are read from one mRNA and different proteins are synthesized. The advantage of this method is that it provides quantitative estimates of ribosome coverage of mRNA and can detect relatively rare translation events. Using this technology, it was possible to identify and classify plant genes by the type of regulation of their expression at the transcription, translation, or both levels. Features of the mRNA structure that affect translation levels have been revealed: the formation of G2 quadruplexes and the presence of specific motifs in the 5’-UTR region, GC content, the presence of alternative translation starts, and the influence of uORFs on the translation of downstream mORFs. In this review, we briefly reviewed the RP methodology and the prospects for its application to study the structural and functional organization and regulation of plant gene expression.

scholarly journals OpenProt 2021: deeper functional annotation of the coding potential of eukaryotic genomes

2020 ◽  
Vol 49 (D1) ◽  
pp. D380-D388 ◽  
Author(s):  
Marie A Brunet ◽  
Jean-François Lucier ◽  
Maxime Levesque ◽  
Sébastien Leblanc ◽  
Jean-Francois Jacques ◽  
...  
Keyword(s):  
Confidence Score ◽  
Ribosome Profiling ◽  
Open Reading Frames ◽  
Supporting Evidence ◽  
Initial Release ◽  
Ncbi Refseq ◽  
Computational Resources ◽  
Analysis Platform ◽  
Eukaryotic Genomes ◽  
Reading Frames

Abstract OpenProt (www.openprot.org) is the first proteogenomic resource supporting a polycistronic annotation model for eukaryotic genomes. It provides a deeper annotation of open reading frames (ORFs) while mining experimental data for supporting evidence using cutting-edge algorithms. This update presents the major improvements since the initial release of OpenProt. All species support recent NCBI RefSeq and Ensembl annotations, with changes in annotations being reported in OpenProt. Using the 131 ribosome profiling datasets re-analysed by OpenProt to date, non-AUG initiation starts are reported alongside a confidence score of the initiating codon. From the 177 mass spectrometry datasets re-analysed by OpenProt to date, the unicity of the detected peptides is controlled at each implementation. Furthermore, to guide the users, detectability statistics and protein relationships (isoforms) are now reported for each protein. Finally, to foster access to deeper ORF annotation independently of one’s bioinformatics skills or computational resources, OpenProt now offers a data analysis platform. Users can submit their dataset for analysis and receive the results from the analysis by OpenProt. All data on OpenProt are freely available and downloadable for each species, the release-based format ensuring a continuous access to the data. Thus, OpenProt enables a more comprehensive annotation of eukaryotic genomes and fosters functional proteomic discoveries.

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 Actively translated uORFs reduce translation and mRNA stability independent of NMD in Arabidopsis

2021 ◽  
Author(s):  
Hsin-Yen Larry Wu ◽  
Polly Yingshan Hsu
Keyword(s):  
Gene Expression ◽  
Mrna Stability ◽  
Gene Expression Regulation ◽  
Regulatory Elements ◽  
Expression Regulation ◽  
Ribosome Profiling ◽  
Translation Efficiency ◽  
Nonsense Mediated Decay ◽  
Protein Levels ◽  
Eukaryotic Genes

ABSTRACTUpstream ORFs (uORFs) are widespread cis-regulatory elements in the 5’ untranslated regions of eukaryotic genes. Translation of uORFs could negatively regulate protein synthesis by repressing main ORF (mORF) translation and by reducing mRNA stability presumably through nonsense-mediated decay (NMD). While the above expectations were supported in animals, they have not been extensively tested in plants. Using ribosome profiling, we systematically identified 2093 Actively Translated uORFs (ATuORFs) in Arabidopsis seedlings and examined their roles in gene expression regulation by integrating multiple genome-wide datasets. Compared with genes without uORFs, we found ATuORFs result in 38%, 14%, and 43% reductions in translation efficiency, mRNA stability, and protein levels, respectively. The effects of predicted but not actively translated uORFs are much weaker than those of ATuORFs. Interestingly, ATuORF-containing genes are also expressed at higher levels and encode longer proteins with conserved domains, features that are common in evolutionarily older genes. Moreover, we provide evidence that uORF translation in plants, unlike in vertebrates, generally does not trigger NMD. We found ATuORF-containing transcripts are degraded through 5’ to 3’ decay, while NMD targets are degraded through both 5’ to 3’ and 3’ to 5’ decay, suggesting uORF-associated mRNA decay and NMD have distinct genetic requirements. Furthermore, we showed ATuORFs and NMD repress translation through separate mechanisms. Our results reveal that the potent inhibition of uORFs on mORF translation and mRNA stability in plants are independent of NMD, highlighting a fundamental difference in gene expression regulation by uORFs in the plant and animal kingdoms.

scholarly journals The Genome Sequence of the Gram-Positive Sugarcane Pathogen Leifsonia xyli subsp. xyli

2004 ◽  
Vol 17 (8) ◽  
pp. 827-836 ◽  
Author(s):  
Claudia B. Monteiro-Vitorello ◽  
Luis E. A. Camargo ◽  
Marie A. Van Sluys ◽  
João P. Kitajima ◽  
Daniela Truffi ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Gc Content ◽  
Open Reading Frames ◽  
Circular Chromosome ◽  
Free Sugars ◽  
Sugar Transporters ◽  
Living Organisms ◽  
Bacterial Plant Pathogen ◽  
Single Circular Chromosome ◽  
Reading Frames

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.

scholarly journals Characterization and Genome Sequence of Mycobacterium Phage XianYue

2020 ◽  
Vol 9 (25) ◽  
Author(s):  
Wesley D. Rhinehart ◽  
Amanda J. Laidlaw ◽  
Alexis M. O’Neal ◽  
Jessica A. Toller ◽  
Miriam Segura-Totten ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Gc Content ◽  
Open Reading Frames ◽  
Nucleotide Identity ◽  
Content Type ◽  
Reading Frames

ABSTRACT Novel mycobacteriophage XianYue was isolated in Northeast Georgia and infects Mycobacteria smegmatis mc2155. Actinobacteriophages which share at least 50% nucleotide identity are grouped into clusters, with XianYue in cluster A2. Its genome is 52,907 bp with 91 open reading frames (ORFs) and 62.9% GC content, and it shares 86.51% nucleotide identity with mycobacteriophage Trixie.

scholarly journals Whole Genome Sequencing and Analysis of Chlorimuron-Ethyl Degrading Bacteria Klebsiella pneumoniae 2N3

2019 ◽  
Vol 20 (12) ◽  
pp. 3053 ◽  
Author(s):  
Cheng Zhang ◽  
Qingkai Hao ◽  
Zhengyi Zhang ◽  
Xianghui Zhang ◽  
Hongyu Pan ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Gene Knockout ◽  
Scientific Evidence ◽  
Gc Content ◽  
Open Reading Frames ◽  
Degradation Rates ◽  
Degrading Bacteria ◽  
High Throughput Dna Sequencing ◽  
Chlorimuron Ethyl ◽  
Reading Frames

Klebsiella pneumoniae 2N3 is a strain of gram-negative bacteria that can degrade chlorimuron-ethyl and grow with chlorimuron-ethyl as the sole nitrogen source. The complete genome of Klebsiella pneumoniae 2N3 was sequenced using third generation high-throughput DNA sequencing technology. The genomic size of strain 2N3 was 5.32 Mb with a GC content of 57.33% and a total of 5156 coding genes and 112 non-coding RNAs predicted. Two hydrolases expressed by open reading frames (ORFs) 0934 and 0492 were predicted and experimentally confirmed by gene knockout to be involved in the degradation of chlorimuron-ethyl. Strains of ΔORF 0934, ΔORF 0492, and wild type (WT) reached their highest growth rates after 8–10 hours in incubation. The degradation rates of chlorimuron-ethyl by both ΔORF 0934 and ΔORF 0492 decreased in comparison to the WT during the first 8 hours in culture by 25.60% and 24.74%, respectively, while strains ΔORF 0934, ΔORF 0492, and the WT reached the highest degradation rates of chlorimuron-ethyl in 36 hours of 74.56%, 90.53%, and 95.06%, respectively. This study provides scientific evidence to support the application of Klebsiella pneumoniae 2N3 in bioremediation to control environmental pollution.

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