Biology subjects

Dedon, P.

Publications and source records attributed to Dedon, P..

9 recordsLinked to original sources

bioRxiv2024

Angiogenin regulates mitochondrial stress and function via tRNA-derived fragments generation and impacting tRNA modifications.

Mitochondrial stress and dysfunction play an important role in many diseases, such as cancer, diabetes, and neurodegenerative diseases. We previously observed that mitochondrial electron transport chain (ETC) inhibition can induce tRNA cleavage and tsRNAs (tRNA-derived small non-coding RNAs) generation. However, whether this process is mediated via Angiogenin (ANG), the canonical enzyme responsible for tRNA cleavage, and whether it has a role in regulating the mitochondrial stress response remains to be understood. ANG is linked to Amyotrophic Lateral Sclerosis (ALS) and other conditions where mitochondrial stress plays a role in pathophysiology. Here, we aimed to examine the role of ANG in regulating the translational response to mitochondrial stress. We observed that ANG protected the cells from respiratory complex III and V inhibition specifically. Furthermore, we validated that the tsRNAs generated during mitochondrial and oxidative stress are mediated by ANG, given that their production is abrogated after ANG knock-out (KO). In addition, we observed that ANG-KO altered the tRNA modification status. Namely, we observed that ANG-KO led to the downregulation of queuosine tRNA modifications (tRNA-Q). tRNA-Q itself is related to mitochondrial translation and function. Indeed, we observed that ANG-KO led to reduced mitochondrial respiration and function. ANG altered how the cells respond to mitochondrial stress by altering the dynamic tRNA modification changes occurring during the stress response. We further examined the impact of ANG-KO on stress granules (SG) assembly as well as the knockdown of G3BP1 (core protein of SGs) on tsRNAs generation. Our results indicate that ANG regulates mitochondrial function and stress via tsRNAs generation as well as altering tRNA modifications levels. Our data also indicate that there are no direct links between tRNA cleavage and SG assembly, and both could be parallel systems for translation repression during stress.

molecular biology↗

bioRxiv2024

Translational response to mitochondrial stresses is orchestrated by tRNA modifications.

Mitochondrial stress and dysfunction play important roles in many pathologies. However, how cells respond to mitochondrial stress is not fully understood. Here, we examined the translational response to electron transport chain (ETC) inhibition and arsenite induced mitochondrial stresses. Our analysis revealed that during mitochondrial stress, tRNA modifications (namely f5C, hm5C, queuosine and its derivatives, and mcm5U) dynamically change to fine tune codon decoding, usage, and optimality. These changes in codon optimality drive the translation of many pathways and gene sets, such as the ATF4 pathway and selenoproteins, involved in the cellular response to mitochondrial stress. We further examined several of these modifications using targeted approaches. ALKBH1 knockout (KO) abrogated f5C and hm5C levels and led to mitochondrial dysfunction, reduced proliferation, and impacted mRNA translation rates. Our analysis revealed that tRNA queuosine (tRNA-Q) is a master regulator of the mitochondrial stress response. KO of QTRT1 or QTRT2, the enzymes responsible for tRNA-Q synthesis, led to mitochondrial dysfunction, translational dysregulation, and metabolic alterations in mitochondria-related pathways, without altering cellular proliferation. In addition, our analysis revealed that tRNA-Q loss led to a domino effect on various tRNA modifications. Some of these changes could be explained by metabolic profiling. Our analysis also revealed that utilizing serum deprivation or alteration with Queuine supplementation to study tRNA-Q or stress response can introduce various confounding factors by altering many other tRNA modifications. In summary, our data show that tRNA modifications are master regulators of the mitochondrial stress response by driving changes in codon decoding.

molecular biology↗

bioRxiv2024

Mapping the tRNA Modification Landscape of Bartonella henselae Houston I and Bartonella quintana Toulouse

Transfer RNA (tRNA) modifications play a crucial role in maintaining translational fidelity and efficiency, and they may function as regulatory elements in stress response and virulence. Despite their pivotal roles, a comprehensive mapping of tRNA modifications and their associated synthesis genes is still limited, with a predominant focus on free-living bacteria. In this study, we employed a multidisciplinary approach, incorporating comparative genomics, mass spectrometry, and next-generation sequencing, to predict the set of tRNA modification genes responsible for tRNA maturation in two intracellular pathogens--Bartonella henselae Houston I and Bartonella quintana Toulouse, which are causative agents of cat-scratch disease and trench fever, respectively. This analysis presented challenges, particularly because of host RNA contamination, which served as a potential source of error. However, our approach predicted 26 genes responsible for synthesizing 23 distinct tRNA modifications in B. henselae and 22 genes associated with 23 modifications in B. quintana. Notably, akin to other intracellular and symbiotic bacteria, both Bartonella species have undergone substantial reductions in tRNA modification genes, mostly by simplifying the hypermodifications present at positions 34 and 37. B. quintana exhibited the additional loss of four modifications and these were linked to examples of gene decay, providing snapshots of reductive evolution.

microbiology↗

bioRxiv2023

Alternate routes to mnm5s2U synthesis in Gram-positive bacteria

The wobble bases of tRNAs that decode split codons are often heavily modified. In Bacteria tRNAGlu, Gln, Asp contain a variety of xnm5s2U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negative Escherichia coli K12 model. Despite the ubiquitous presence of mnm5s2U modification, genomic analysis shows the absence of mnmC orthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the installation of this modification. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the Radical Sam superfamily was found to be involved in the synthesis of mnm5s2U in both Bacillus subtilis and Streptococcus mutans. This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm5s2U into mnm5s2U in B. subtilis. Analysis of tRNA modifications of both S. mutans and Streptococcus pneumoniae shows that growth conditions and genetic backgrounds influence the ratios of pathways intermediates in regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. The occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in nature. ImportanceThe xnm5s2U modifications found in several tRNAs at the wobble base position are widespread in Bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile Radical SAM superfamily and is involved in the synthesis of mnm5s2U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications.

microbiology↗

bioRxiv2023

Mammalian tissue specific translation regulation; role of tRNA epitranscriptome in regulating codon optimality patterns across tissues.

The tRNA epitranscriptome has been recognized as an important player in mRNA translation regulation. Our knowledge of the role of tRNA epitranscriptome in fine-tuning translation codon decoding at tissue or cell levels remains incomplete. Here, we analyzed seven tissues from mice for the expression of tRNA modifications and mature tRNAs as well as mRNA translation and codon decoding. Our analysis revealed distinct enrichment patterns of tRNA modifications in tissues. Queuosine (Q) tRNA modification was most enriched in the brain compared to other tissues, while mitochondrial tRNA modifications and tRNA expression was highest in the heart. Using three different metrics for codon analysis; isoacceptors frequencies, total codon frequencies, and A-site pausing, we revealed a strong bias towards A/T ending codons in most tissues except for the brain. Using this observation, we synthesized, and delivered in vivo, codon mutated EGFP for Q-codons, where the C-ending Q-codons were replaced with U-ending codons. The protein levels of mutant EGFP were downregulated in liver, which is poor in Q, when NAC codons were exchanged for NAU codons, while in brain EGFP levels did not change. This data shows that understanding tRNA modifications enrichments across tissues is not only essential for understanding codon decoding and bias, but it can also be utilized for optimizing gene and mRNA therapeutics to be more tissue, cell, or condition specific.

molecular biology↗

bioRxiv2023

Accumulation of m6A exhibits stronger correlation with MAPT than beta-amyloid pathology in an APP NL-G-F /MAPT P301S mouse model of Alzheimer's disease

The study for the pathophysiology study of Alzheimers disease (AD) has been hampered by lack animal models that recapitulate the major AD pathologies, including extracellular {beta}-amyloid (A{beta}) deposition, intracellular aggregation of microtubule associated protein tau (MAPT), inflammation and neurodegeneration. We now report on a double transgenic APPNL-G-F MAPTP301S mouse that at 6 months of age exhibits robust A{beta} plaque accumulation, intense MAPT pathology, strong inflammation and extensive neurodegeneration. The presence of A{beta} pathology potentiated the other major pathologies, including MAPT pathology, inflammation and neurodegeneration. However, MAPT pathology neither changed levels of amyloid precursor protein nor potentiated A{beta} accumulation. The APPNL-G-F/MAPTP301S mouse model also showed strong accumulation of N6-methyladenosine (m6A), which was recently shown to be elevated in the AD brain. M6A primarily accumulated in neuronal soma, but also co-localized with a subset of astrocytes and microglia. The accumulation of m6A corresponded with increases in METTL3 and decreases in ALKBH5, which are enzymes that add or remove m6A from mRNA, respectively. Thus, the APPNL- G-F/MAPTP301S mouse recapitulates many features of AD pathology beginning at 6 months of aging.

neuroscience↗

bioRxiv2022

An RNA modification enzyme directly senses reactive oxygen species for translational regulation in Enterococcus faecalis

Bacteria possess elaborate systems to manage reactive oxygen and nitrogen species (ROS) arising from exposure to the mammalian immune system and environmental stresses. Here we report the discovery of an ROS-sensing RNA-modifying enzyme that regulates translation of stress-response proteins in the gut commensal and opportunistic pathogen Enterococcus faecalis. We analyzed the tRNA epitranscriptome of E. faecalis in response to reactive oxygen species (ROS) or sublethal doses of ROS-inducing antibiotics and identified large decreases in N2-methyladenosine (m2A) in both 23S ribosomal RNA and transfer RNA. This we determined to be due to ROS-mediated inactivation of the Fe-S cluster-containing methyltransferase, RlmN. Genetic knockout of RlmN gave rise to a proteome that mimicked the oxidative stress response, with increased levels of superoxide dismutase and decreased virulence proteins. While tRNA modifications are established to be dynamic for fine-tuning translation, here we report the first instance of a dynamically regulated, environmentally responsive rRNA modification. These studies lead to model in which RlmN serves as a redox-sensitive molecular switch, directly relaying oxidative stress to modulating translation through the rRNA and the tRNA epitranscriptome, revealing a new paradigm for understanding direct regulation of the proteome by RNA modifications.

microbiology↗

bioRxiv2022

DNA Modifications Enabling Proximity Biotinylation

Advances in peroxidase- and biotin ligase-mediated signal amplification have enabled high-resolution subcellular mapping of endogenous RNA localization and protein-protein interactions. Application of these technologies has been limited to RNA and proteins because of the reactive groups required for biotinylation in each context. Here we report several novel methods for proximity biotinylation of exogenous oligodeoxyribonucleotides by application of well-established and convenient enzymatic tools. We describe approaches using simple and efficient conjugation chemistries to modify deoxyribonucleotides with "antennae" sensitive to phenoxy radical or biotinoyl-5-adenylate. In addition, we report chemical details of a previously undescribed adduct between tryptophan and a phenoxy radical group. These developments have potential application in the selection of exogenous nucleic acids capable of unaided entry into living cells.

biochemistry↗

bioRxiv2022

Queuine salvaging in the human parasite Entamoeba histolytica

Queuosine (Q) is a naturally occurring modified nucleoside that occurs in the first position of transfer RNA anticodons such as Asp, Asn, His, and Tyr. As eukaryotes lack pathways to synthesize queuine, the Q nucleobase, they must obtain it from their diet or gut microbiota. Previously, we described the effects of queuine on the physiology of the eukaryotic parasite Entamoeba histolytica and characterized the enzyme EhTGT responsible for queuine incorporation into tRNA. At present, it is unknown how E. histolytica salvages Q from gut bacteria. We used liquid chromatography-mass spectrometry (LC-MS) and N-acryloyl-3-aminophenylboronic acid (APB) PAGE analysis to demonstrate that E. histolytica trophozoites can salvage queuine from Q or E. coli K12 but not from the modified E. coli QueC strain, which cannot produce queuine. We then examined the role of EhDUF2419, a protein with homology to DNA glycosylase, as queuine salvage enzyme in E. histolytica. We found that glutathione S-transferase (GST)-EhDUF2419 catalyzed the conversion of Q into queuine. Trophozoites silenced for EhDUF2419 expression are impaired in their ability to form Q-tRNA from Q or from E. coli. We also observed that Q partially protects control trophozoites from oxidative stress (OS), but not siEhDUF2419 trophozoites. Overall, our data reveal that EhDUF2419 is central for the salvaging of queuine from bacteria and for the resistance of the parasite to OS.

microbiology↗