Genome recoding by tRNA modifications

RNA modifications are emerging as an additional regulatory layer on top of the primary RNA sequence. These modifications are particularly enriched in tRNAs where they can regulate not only global protein translation, but also protein translation at the codon level. Modifications located in or in the vicinity of tRNA anticodons are highly conserved in eukaryotes and have been identified as potential regulators of mRNA decoding. Recent studies have provided novel insights into how these modifications orchestrate the speed and fidelity of translation to ensure proper protein homeostasis. This review highlights the prominent modifications in the tRNA anticodon loop: queuosine, inosine, 5-methoxycarbonylmethyl-2-thiouridine, wybutosine, threonyl–carbamoyl–adenosine and 5-methylcytosine. We discuss the functional relevance of these modifications in protein translation and their emerging role in eukaryotic genome recoding during cellular adaptation and disease.

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Bacterial tRNA 2′-O-methylation is dynamically regulated under stress conditions and modulates innate immune response

Nucleic Acids Research ◽

10.1093/nar/gkaa1123 ◽

2020 ◽

Vol 48 (22) ◽

pp. 12833-12844

Author(s):

Adeline Galvanin ◽

Lea-Marie Vogt ◽

Antonia Grober ◽

Isabel Freund ◽

Lilia Ayadi ◽

...

Keyword(s):

Gene Expression Regulation ◽

Current Knowledge ◽

Protein Translation ◽

Stress Conditions ◽

Transcriptional Level ◽

Trna Modification ◽

Rna Modifications ◽

Swarming Motility ◽

E Coli ◽

Response To Stress

Abstract RNA modifications are a well-recognized way of gene expression regulation at the post-transcriptional level. Despite the importance of this level of regulation, current knowledge on modulation of tRNA modification status in response to stress conditions is far from being complete. While it is widely accepted that tRNA modifications are rather dynamic, such variations are mostly assessed in terms of total tRNA, with only a few instances where changes could be traced to single isoacceptor species. Using Escherichia coli as a model system, we explored stress-induced modulation of 2′-O-methylations in tRNAs by RiboMethSeq. This analysis and orthogonal analytical measurements by LC-MS show substantial, but not uniform, increase of the Gm18 level in selected tRNAs under mild bacteriostatic antibiotic stress, while other Nm modifications remain relatively constant. The absence of Gm18 modification in tRNAs leads to moderate alterations in E. coli mRNA transcriptome, but does not affect polysomal association of mRNAs. Interestingly, the subset of motility/chemiotaxis genes is significantly overexpressed in ΔTrmH mutant, this corroborates with increased swarming motility of the mutant strain. The stress-induced increase of tRNA Gm18 level, in turn, reduced immunostimulation properties of bacterial tRNAs, which is concordant with the previous observation that Gm18 is a suppressor of Toll-like receptor 7 (TLR7)-mediated interferon release. This documents an effect of stress induced modulation of tRNA modification that acts outside protein translation.

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Regulatory roles of RNA modifications in breast cancer

NAR Cancer ◽

10.1093/narcan/zcab036 ◽

2021 ◽

Vol 3 (3) ◽

Author(s):

Kanchan Kumari ◽

Paula Groza ◽

Francesca Aguilo

Keyword(s):

Breast Cancer ◽

Messenger Rna ◽

Therapeutic Strategies ◽

Rna Modifications ◽

Breast Tumorigenesis ◽

Cellular Processes ◽

Trna Modifications ◽

Expression Control ◽

Gene Expression Control ◽

Ribosomal Rrna

Abstract Collectively referred to as the epitranscriptome, RNA modifications play important roles in gene expression control regulating relevant cellular processes. In the last few decades, growing numbers of RNA modifications have been identified not only in abundant ribosomal (rRNA) and transfer RNA (tRNA) but also in messenger RNA (mRNA). In addition, many writers, erasers and readers that dynamically regulate the chemical marks have also been characterized. Correct deposition of RNA modifications is prerequisite for cellular homeostasis, and its alteration results in aberrant transcriptional programs that dictate human disease, including breast cancer, the most frequent female malignancy, and the leading cause of cancer-related death in women. In this review, we emphasize the major RNA modifications that are present in tRNA, rRNA and mRNA. We have categorized breast cancer-associated chemical marks and summarize their contribution to breast tumorigenesis. In addition, we describe less abundant tRNA modifications with related pathways implicated in breast cancer. Finally, we discuss current limitations and perspectives on epitranscriptomics for use in therapeutic strategies against breast and other cancers.

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Crosstalk of lipid and protein homeostasis to maintain membrane function

Biological Chemistry ◽

10.1515/hsz-2013-0235 ◽

2014 ◽

Vol 395 (3) ◽

pp. 313-326 ◽

Cited By ~ 15

Author(s):

Claudius Stordeur ◽

Kristina Puth ◽

James P. Sáenz ◽

Robert Ernst

Keyword(s):

Protein Quality ◽

Lipid Homeostasis ◽

Protein Homeostasis ◽

Membrane Biogenesis ◽

Membrane Function ◽

Biochemical Processes ◽

Cellular Life ◽

Selective Diffusion ◽

Functional Relevance ◽

Droplet Morphology

Abstract Biological membranes are a defining feature of cellular life. They serve as selective diffusion barriers, compartmentalize biochemical processes and protect the cellular milieu. We are only beginning to understand the principles underlying their homeostasis and the functional relevance of their complex compositions. Here, we summarize some recent evidences that suggest an intense crosstalk between the pathways of protein quality control and lipid homeostasis. We discuss paradigms of lipid regulation by protein degradation machineries and highlight the intricate connections between lipid droplet morphology, membrane biogenesis and ER-stress.

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A Census and Categorization Method of Epitranscriptomic Marks

International Journal of Molecular Sciences ◽

10.3390/ijms21134684 ◽

2020 ◽

Vol 21 (13) ◽

pp. 4684

Author(s):

Julia Mathlin ◽

Loredana Le Pera ◽

Teresa Colombo

Keyword(s):

Gene Expression ◽

Rna Binding ◽

Rna Binding Proteins ◽

Current Knowledge ◽

Messenger Rnas ◽

Rna Modifications ◽

Chemical Modifications ◽

Rna Molecules ◽

Transcriptional Regulatory ◽

Functional Relevance

In the past few years, thorough investigation of chemical modifications operated in the cells on ribonucleic acid (RNA) molecules is gaining momentum. This new field of research has been dubbed “epitranscriptomics”, in analogy to best-known epigenomics, to stress the potential of ensembles of RNA modifications to constitute a post-transcriptional regulatory layer of gene expression orchestrated by writer, reader, and eraser RNA-binding proteins (RBPs). In fact, epitranscriptomics aims at identifying and characterizing all functionally relevant changes involving both non-substitutional chemical modifications and editing events made to the transcriptome. Indeed, several types of RNA modifications that impact gene expression have been reported so far in different species of cellular RNAs, including ribosomal RNAs, transfer RNAs, small nuclear RNAs, messenger RNAs, and long non-coding RNAs. Supporting functional relevance of this largely unknown regulatory mechanism, several human diseases have been associated directly to RNA modifications or to RBPs that may play as effectors of epitranscriptomic marks. However, an exhaustive epitranscriptome’s characterization, aimed to systematically classify all RNA modifications and clarify rules, actors, and outcomes of this promising regulatory code, is currently not available, mainly hampered by lack of suitable detecting technologies. This is an unfortunate limitation that, thanks to an unprecedented pace of technological advancements especially in the sequencing technology field, is likely to be overcome soon. Here, we review the current knowledge on epitranscriptomic marks and propose a categorization method based on the reference ribonucleotide and its rounds of modifications (“stages”) until reaching the given modified form. We believe that this classification scheme can be useful to coherently organize the expanding number of discovered RNA modifications.

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Abundances of transfer RNA modifications and transcriptional levels for tRNA-modifying enzymes are sex-specific in mosquitoes

10.1101/2021.08.03.454936 ◽

2021 ◽

Author(s):

Melissa Kelley ◽

Melissa R Uhran ◽

Cassandra Herbert ◽

George Yoshida ◽

Emmarie Watts ◽

...

Keyword(s):

Mosquito Species ◽

Critical Factor ◽

Transfer Rna ◽

Trna Modification ◽

Rna Modifications ◽

Transcript Levels ◽

Trna Modifications ◽

Remediation Strategies ◽

Adapter Molecule ◽

Novel Target

As carriers of multiple human diseases, understanding the mechanisms behind mosquito reproduction may have implications for remediation strategies. Transfer RNA (tRNA) acts as the adapter molecule of amino acids and are key components in protein synthesis and a critical factor in the function of tRNAs is chemical modifications. Here, we provide an assessment of tRNA modifications between sexes for three mosquito species and examine correlated transcript levels underlying key proteins involved in tRNA modification. Thirty-three tRNA modifications were detected among mosquito species and most of these modifications are higher in females compared to males. Analysis of previous male and female RNAseq datasets indicated a similar increase in tRNA modifying enzymes in females, supporting our observed female enrichment of tRNA modifications. Tissues-specific expressional studies revealed high transcript levels for tRNA modifying enzymes in the ovaries for Aedes aegypti, but not male reproductive tissues. These studies suggest that tRNA modifications may be critical to reproduction in mosquitoes, representing a potential novel target for control.

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Identification of a motif in Trm732 required for 2′-O-methylation of the tRNA anticodon loop by Trm7

10.1101/2021.06.03.446962 ◽

2021 ◽

Author(s):

Holly M. Funk ◽

Daisy J. DiVita ◽

Hannah E. Sizemore ◽

Kendal Wehrle ◽

Catherine L. Weiner ◽

...

Keyword(s):

Yeast Cells ◽

Trna Modification ◽

Anticodon Loop ◽

Secondary Effects ◽

Cellular Functions ◽

Trna Modifications ◽

Modification Activity ◽

Human Disorders ◽

Methylation Activity

Posttranscriptional tRNA modifications are essential for proper gene expression, and defects in the enzymes that perform tRNA modifications are associated with numerous human disorders. Throughout eukaryotes, 2′-O-methylation of residues 32 and 34 of the anticodon loop of tRNA is important for proper translation, and in humans, lack of these modifications results in non-syndromic X-linked intellectual disability. In yeast, the methyltransferase Trm7 forms a complex with Trm732 to 2′-O-methylate tRNA residue 32 and with Trm734 to 2′-O-methylate tRNA residue 34. Trm732 and Trm734 are required for the methylation activity of Trm7, but the role of these auxiliary proteins is not clear. Additionally, Trm732 and Trm734 homologs are implicated in biological processes not directly related to translation, suggesting that these proteins may have additional cellular functions. To identify critical amino acids in Trm732, we generated variants and tested their ability to function in yeast cells. We identified a conserved RRSAGLP motif in the conserved DUF2428 domain of Trm732 that is required for tRNA modification activity by both yeast Trm732 and its human homolog THADA. The identification of Trm732 variants that lack tRNA modification activity will help to determine if other biological functions ascribed to Trm732 and THADA are directly due to tRNA modification, or to secondary effects due to other functions of these proteins.

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MLC-Seq: de novo Sequencing of Full-Length tRNAs and Quantitative Mapping of Multiple RNA Modifications

10.21203/rs.3.rs-1090754/v1 ◽

2021 ◽

Author(s):

Shenglong Zhang ◽

Xiaohong Yuan ◽

Yue Su ◽

Xudong Zhang ◽

Spencer Turkel ◽

...

Keyword(s):

Mass Spectrometry ◽

De Novo ◽

Direct Sequencing ◽

Rna Modifications ◽

Rna Sequences ◽

Trna Modifications ◽

Quantitative Mapping ◽

Sequencing Method ◽

Direct Sequencing Method ◽

Generation Sequencing

Abstract Despite the extensive use of next-generation sequencing of RNA, simultaneous sequencing and quantitative mapping of multiple RNA modifications remain challenging. Herein, we develop MLC-Seq, a mass spectrometry-based direct sequencing method allowing for simultaneously unravelling the RNA sequences and quantitatively mapping different tRNA nucleotide modifications site-specifically. Importantly, MLC-Seq reveals the stoichiometric changes of tRNA modifications upon treatment with the dealkylating enzyme AlkB, and led to the discovery of new nucleotide modifications.

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A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057115623264 ◽

2015 ◽

Vol 21 (3) ◽

pp. 290-297 ◽

Cited By ~ 18

Author(s):

Fengling Li ◽

Steven Kennedy ◽

Taraneh Hajian ◽

Elisa Gibson ◽

Alma Seitova ◽

...

Keyword(s):

Protein Translation ◽

Export Control ◽

Protein Splicing ◽

Rna Modifications ◽

Cellular Processes ◽

Nuclear Rna ◽

Rna Export ◽

Nuclear Rna Export ◽

Assay Conditions

N6-methyladenosine (m6A) is the most common reversible internal modification in mammalian RNA. Changes in m6A levels have been implicated in a variety of cellular processes, including nuclear RNA export, control of protein translation, and protein splicing, and they have been linked to obesity, cancer, and other human diseases. METTL3 and METTL14 are N6-adenosine methyltransferases that work more efficiently in a stable METTL3-METTL14 heterodimer complex (METTL3-14). ALKBH5 is an m6A-RNA demethylase that belongs to the AlkB family of dioxygenases. We report the development of radioactivity-based assays for kinetic characterization of m6A-RNA modifications by METTL3-14 complex and ALKBH5 and provide optimal assay conditions suitable for screening for ligands in a 384-well format with Z′ factors of 0.78 and 0.77, respectively.

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Unfolded Protein Response Suppression in Yeast by Loss of tRNA Modifications

Genes ◽

10.3390/genes9110516 ◽

2018 ◽

Vol 9 (11) ◽

pp. 516 ◽

Cited By ~ 5

Author(s):

Alexander Bruch ◽

Roland Klassen ◽

Raffael Schaffrath

Keyword(s):

Unfolded Protein Response ◽

Protein Homeostasis ◽

Anticodon Loop ◽

Transfer Rnas ◽

Unfolded Protein ◽

Optimal Codon ◽

Trna Species ◽

The Unfolded Protein Response ◽

Protein Response ◽

Yeast Mutants

Modifications in the anticodon loop of transfer RNAs (tRNAs) have been shown to ensure optimal codon translation rates and prevent protein homeostasis defects that arise in response to translational pausing. Consequently, several yeast mutants lacking important anticodon loop modifications were shown to accumulate protein aggregates. Here we analyze whether this includes the activation of the unfolded protein response (UPR), which is commonly triggered by protein aggregation within the endoplasmic reticulum (ER). We demonstrate that two different aggregation prone tRNA modification mutants (elp6 ncs2; elp3 deg1) lacking combinations of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U: elp3; elp6; ncs2) and pseudouridine (Ψ: deg1) reduce, rather than increase, splicing of HAC1 mRNA, an event normally occurring as a precondition of UPR induction. In addition, tunicamycin (TM) induced HAC1 splicing is strongly impaired in the elp3 deg1 mutant. Strikingly, this mutant displays UPR independent resistance against TM, a phenotype we found to be rescued by overexpression of tRNAGln(UUG), the tRNA species usually carrying the mcm5s2U34 and Ψ38 modifications. Our data indicate that proper tRNA anticodon loop modifications promote rather than impair UPR activation and reveal that protein synthesis and homeostasis defects in their absence do not routinely result in UPR induction but may relieve endogenous ER stress.

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Quantitative tRNA-sequencing uncovers metazoan tissue-specific tRNA regulation

10.21203/rs.3.pex-1031/v1 ◽

2021 ◽

Author(s):

Otis Pinkard ◽

Sean Mcfarland ◽

Thomas Sweet ◽

Jeff Coller

Keyword(s):

Messenger Rna ◽

Protein Production ◽

Rna Stability ◽

Amino Acid Identity ◽

Rna Modifications ◽

Transfer Rnas ◽

Trna Modifications ◽

Mammalian Tissues ◽

Trna Sequencing ◽

Regulatory Landscape

Abstract Transfer RNAs (tRNA) are quintessential in deciphering the genetic code; disseminating nucleic acid triplets into correct amino acid identity. While this decoding function is clear, an emerging theme is that tRNA abundance and functionality can powerfully impact protein production rate, folding, activity, and messenger RNA stability. Importantly, however, the expression pattern of tRNAs is obliquely known. Here we present Quantitative Mature tRNA sequencing (QuantM-tRNA seq), a technique to monitor tRNA abundance and sequence variants secondary to RNA modifications. With QuantM-tRNA seq we assess the tRNA transcriptome in mammalian tissues. We observe dramatic distinctions in isodecoder expression and known tRNA modifications between tissues. Remarkably, despite dramatic changes in tRNA isodecoder gene expression, the overall anticodon pool of each tRNA family is similar across tissues. These findings suggest that while anticodon pools appear to be buffered via an unknown mechanism, underlying transcriptomic and epitranscriptomic differences suggest a more complex tRNA regulatory landscape.

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