Ribosome Profiling and RNA Sequencing Reveal Genome-Wide Cellular Translation and Transcription Regulation Under Osmotic Stress in Lactobacillus rhamnosus ATCC 53103

To determine whether osmotic pressure affects the translation efficiency of Lactobacillus rhamnosus, the ribosome profiling assay was performed to analyze the changes in translation efficiency in L. rhamnosus ATCC 53103. Under osmotic stress, differentially expressed genes (DEGs) involved in fatty acid biosynthesis and metabolism, ribosome, and purine metabolism pathways were co-regulated with consistent expression direction at translation and transcription levels. DEGs involved in the biosynthesis of phenylalanine, tyrosine, and tryptophan, and the phosphotransferase system pathways also were co-regulated at translation and transcription levels, while they showed opposite expression direction at two levels. Moreover, DEGs involved in the two-component system, amino acid metabolism, and pyruvate metabolism pathways were only regulated at the transcription level. And DEGs involved in fructose and mannose metabolism were only regulated at the translation level. The translation efficiency of DEGs involved in the biosynthesis of amino acids was downregulated while in quorum sensing and PTS pathways was upregulated. In addition, the ribosome footprints accumulated in open reading frame regions resulted in impaired translation initiation and elongation under osmotic stress. In summary, L. rhamnosus ATCC 53103 could respond to osmotic stress by translation regulation and control the balance between survival and growth of cells by transcription and translation.

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RiboDiff: Detecting Changes of Translation Efficiency from Ribosome Footprints

10.1101/017111 ◽

2015 ◽

Cited By ~ 3

Author(s):

Yi Zhong ◽

Theofanis Karaletsos ◽

Philipp Drewe ◽

Vipin Thankam T Sreedharan ◽

David Kuo ◽

...

Keyword(s):

Linear Models ◽

Protein Translation ◽

Ribosome Profiling ◽

Translation Regulation ◽

Analysis Tool ◽

Translation Efficiency ◽

Case Control Studies ◽

Statistical Framework ◽

Experimental Treatments ◽

Over Dispersion

Motivation: Deep sequencing based ribosome footprint profiling can provide novel insights into the regulatory mechanisms of protein translation. However, the observed ribosome profile is fundamentally confounded by transcriptional activity. In order to decipher principles of translation regulation, tools that can reliably detect changes in translation efficiency in case-control studies are needed. Results: We present a statistical framework and analysis tool, RiboDiff, to detect genes with changes in translation efficiency across experimental treatments. RiboDiff uses generalized linear models to estimate the over-dispersion of RNA-Seq and ribosome profiling measurements separately, and performs a statistical test for differential translation efficiency using both mRNA abundance and ribosome occupancy. Availability: Source code and documentation are available at http://github.com/ratschlab/ribodiff. Supplementary Material can be found at http://bioweb.me/ribo.

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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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Loss of Ftsj1 perturbs codon-specific translation efficiency in the brain and is associated with X-linked intellectual disability

Science Advances ◽

10.1126/sciadv.abf3072 ◽

2021 ◽

Vol 7 (13) ◽

pp. eabf3072

Author(s):

Y. Nagayoshi ◽

T. Chujo ◽

S. Hirata ◽

H. Nakatsuka ◽

C.-W. Chen ◽

...

Keyword(s):

Intellectual Disability ◽

State Level ◽

Memory Deficits ◽

Ribosome Profiling ◽

Translation Efficiency ◽

Trna Modification ◽

Synaptic Organization ◽

Transfer Rnas ◽

The Brain

FtsJ RNA 2′-O-methyltransferase 1 (FTSJ1) gene has been implicated in X-linked intellectual disability (XLID), but the molecular pathogenesis is unknown. We show that Ftsj1 is responsible for 2′-O-methylation of 11 species of cytosolic transfer RNAs (tRNAs) at the anticodon region, and these modifications are abolished in Ftsj1 knockout (KO) mice and XLID patient–derived cells. Loss of 2′-O-methylation in Ftsj1 KO mouse selectively reduced the steady-state level of tRNAPhe in the brain, resulting in a slow decoding at Phe codons. Ribosome profiling showed that translation efficiency is significantly reduced in a subset of genes that need to be efficiently translated to support synaptic organization and functions. Ftsj1 KO mice display immature synaptic morphology and aberrant synaptic plasticity, which are associated with anxiety-like and memory deficits. The data illuminate a fundamental role of tRNA modification in the brain through regulation of translation efficiency and provide mechanistic insights into FTSJ1-related XLID.

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m5U54 tRNA Hypomodification by Lack of TRMT2A Drives the Generation of tRNA-Derived Small RNAs

International Journal of Molecular Sciences ◽

10.3390/ijms22062941 ◽

2021 ◽

Vol 22 (6) ◽

pp. 2941

Author(s):

Marisa Pereira ◽

Diana R. Ribeiro ◽

Miguel M. Pinheiro ◽

Margarida Ferreira ◽

Stefanie Kellner ◽

...

Keyword(s):

Stress Response ◽

Small Rnas ◽

Mammalian Cells ◽

Cellular Stress ◽

Cellular Stress Response ◽

Translation Regulation ◽

Translation Efficiency ◽

Down Regulation ◽

Gene Expression Control ◽

The Impact

Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation to gene expression control and cellular stress response. Recent evidence indicates that tsRNAs are also modified, however, the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (m5U) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces m5U54 tRNA hypomodification and tsRNA formation. More specifically, m5U54 hypomodification is followed by overexpression of the ribonuclease angiogenin (ANG) that cleaves tRNAs near the anticodon, resulting in accumulation of 5′tRNA-derived stress-induced RNAs (5′tiRNAs), namely 5′tiRNA-GlyGCC and 5′tiRNA-GluCTC, among others. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently m5U54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tiRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tiRNA formation in mammalian cells. These results establish a link between tRNA hypomethylation and ANG-dependent tsRNAs formation and unravel m5U54 as a tRNA cleavage protective mark.

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Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2704-2711.1986 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2704-2711 ◽

Cited By ~ 1

Author(s):

D S Peabody ◽

S Subramani ◽

P Berg

Keyword(s):

Mammalian Cells ◽

Simian Virus 40 ◽

Simian Virus ◽

Initiation Codon ◽

Efficient Production ◽

Translation Efficiency ◽

Reading Frame ◽

Recombinant Viruses ◽

Internal Translation ◽

Late Region

In a previous report (S. Subramani, R. Mulligan, and P. Berg, Mol. Cell. Biol. 1:854-864, 1981), it was shown that mouse dihydrofolate reductase (DHFR) could be efficiently expressed from simian virus 40 recombinant viruses containing the DHFR cDNA in different locations in the viral late region. This was true even in the case of the SVGT7dhfr26 recombinant, which had the DHFR coding sequence 700 to 800 nucleotides from the 5' end of the mRNA, where it was preceded by the VP2 and VP3 initiator AUGs and a number of other noninitiator AUGs. To investigate the process of internal translation initiation in mammalian cells, we constructed a series of SVGT7dhfr recombinants in which the upstream VP2 and VP3 reading frame was terminated in various positions relative to the DHFR initiation codon. The efficient production of DHFR in infected CV1 cells depended on having the terminators of the VP2-VP3 reading frame positioned upstream or nearby downstream from the DHFR initiation codon. These results reinforce the notion that mammalian ribosomes are capable of translational reinitiation.

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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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Duck Hepatitis A Virus Type 1 Induces eIF2α Phosphorylation-Dependent Cellular Translation Shutoff via PERK/GCN2

Frontiers in Microbiology ◽

10.3389/fmicb.2021.624540 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yuanzhi Liu ◽

Anchun Cheng ◽

Mingshu Wang ◽

Sai Mao ◽

Xumin Ou ◽

...

Keyword(s):

Virus Type ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Translation Efficiency ◽

Eif2α Phosphorylation ◽

Duck Hepatitis ◽

Cellular Translation ◽

First Time

Duck hepatitis A virus type 1 (DHAV-1) is one of the most deadly pathogens that endanger the duck industry. Most viruses usually turn off host translation after infection to facilitate viral replication and translation. For the first time report to our knowledge, DHAV-1 can induce eIF2α phosphorylation and inhibit cellular translation in duck embryo fibroblasts (DEFs). Moreover, the activity of DHAV-1 in the cells caused obvious eIF2α phosphorylation, which has nothing to do with the viral protein. Subsequently, we screened two kinases (PERK and GCN2) that affect eIF2α phosphorylation through inhibitors and shRNA. Notably, the role of GCN2 in other picornaviruses has not been reported. In addition, when the phosphorylation of eIF2α induced by DHAV-1 is inhibited, the translation efficiency of DEFs restores to a normal level, indicating that DHAV-1 induced cellular translation shutoff is dependent on eIF2α phosphorylation.

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Seeking a Role for Translational Control by Alternative Polyadenylation in Saccharomyces cerevisiae

Microorganisms ◽

10.3390/microorganisms9091885 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1885

Author(s):

Rachael E. Turner ◽

Traude H. Beilharz

Keyword(s):

Saccharomyces Cerevisiae ◽

Translational Control ◽

Model Organism ◽

Alternative Polyadenylation ◽

Ribosome Profiling ◽

Translation Efficiency ◽

Mrna Isoforms ◽

Cleavage And Polyadenylation ◽

Made In

Alternative polyadenylation (APA) represents an important mechanism for regulating isoform-specific translation efficiency, stability, and localisation. Though some progress has been made in understanding its consequences in metazoans, the role of APA in the model organism Saccharomyces cerevisiae remains a relative mystery because, despite abundant studies on the translational state of mRNA, none differentiate mRNA isoforms’ alternative 3′-end. This review discusses the implications of alternative polyadenylation in S. cerevisiae using other organisms to draw inferences. Given the foundational role that research in this yeast has played in the discovery of the mechanisms of cleavage and polyadenylation and in the drivers of APA, it is surprising that such an inference is required. However, because advances in ribosome profiling are insensitive to APA, how it impacts translation is still unclear. To bridge the gap between widespread observed APA and the discovery of any functional consequence, we also provide a review of the experimental techniques used to uncover the functional importance of 3′ UTR isoforms on translation.

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

10.1101/2021.09.16.460672 ◽

2021 ◽

Cited By ~ 1

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.

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RiboToolkit: an integrated platform for analysis and annotation of ribosome profiling data to decode mRNA translation at codon resolution

Nucleic Acids Research ◽

10.1093/nar/gkaa395 ◽

2020 ◽

Vol 48 (W1) ◽

pp. W218-W229 ◽

Cited By ~ 1

Author(s):

Qi Liu ◽

Tanya Shvarts ◽

Piotr Sliz ◽

Richard I Gregory

Keyword(s):

Mrna Translation ◽

Ribosome Profiling ◽

Open Reading Frames ◽

Translational Efficiency ◽

Translation Efficiency ◽

Sequencing Data ◽

Experimental Conditions ◽

Web Based ◽

Rna Translation ◽

Web Interfaces

Abstract Ribosome profiling (Ribo-seq) is a powerful technology for globally monitoring RNA translation; ranging from codon occupancy profiling, identification of actively translated open reading frames (ORFs), to the quantification of translational efficiency under various physiological or experimental conditions. However, analyzing and decoding translation information from Ribo-seq data is not trivial. Although there are many existing tools to analyze Ribo-seq data, most of these tools are designed for specific or limited functionalities and an easy-to-use integrated tool to analyze Ribo-seq data is lacking. Fortunately, the small size (26–34 nt) of ribosome protected fragments (RPFs) in Ribo-seq and the relatively small amount of sequencing data greatly facilitates the development of such a web platform, which is easy to manipulate for users with or without bioinformatic expertise. Thus, we developed RiboToolkit (http://rnabioinfor.tch.harvard.edu/RiboToolkit), a convenient, freely available, web-based service to centralize Ribo-seq data analyses, including data cleaning and quality evaluation, expression analysis based on RPFs, codon occupancy, translation efficiency analysis, differential translation analysis, functional annotation, translation metagene analysis, and identification of actively translated ORFs. Besides, easy-to-use web interfaces were developed to facilitate data analysis and intuitively visualize results. Thus, RiboToolkit will greatly facilitate the study of mRNA translation based on ribosome profiling.

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