Quantification of substoichiometric modification reveals global tsRNA hypomodification, preferences for angiogenin-mediated tRNA cleavage, and idiosyncratic epitranscriptomes of human neuronal cell-lines

Pichot, Florian; Hogg, Marion C.; Marchand, Virginie; Bourguignon, Valérie; Jirström, Elisabeth; Farrell, Cliona; Gibriel, Hesham; Prehn, Jochen H M; Motorin, Yuri; Helm, Mark

doi:10.1016/j.csbj.2022.12.020

Cited by 10 publications

(9 citation statements)

References 74 publications

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“…This is understandable given numerous studies that demonstrate modifications can block generation of tRFs by preventing ribonucleases from accessing or acting on their preferred site in the tRNA [20][21][22]53 . This also suggests that tRFs generated in neural cells do not appear to be substrates for de novo methylation, although this was a short experimental time frame 52 .…”

Section: Detection Methodsmentioning

confidence: 97%

“…However, when these techniques are used on total cellular RNA, they can lead to very high reads originating from ribosomal RNAs and tRNAs, as they more accurately reflect the original RNA type distribution (rRNA: ~70 %, tRNA: ~10-15 %). It has recently been shown that tRFs in neural cells are hypomodified indicating demethylation may have little impact on the abundance of tRFs originating from neurons 52 . This is understandable given numerous studies that demonstrate modifications can block generation of tRFs by preventing ribonucleases from accessing or acting on their preferred site in the tRNA [20][21][22]53 .…”

Section: Detection Methodsmentioning

confidence: 99%

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tRNA fragment biomarkers of Neurological Disease: Challenges and Opportunities

Hogg

2023

MRAJ

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Transfer RNAs play a crucial role in protein translation where they bring amino acids to the ribosome to be incorporated into nascent polypeptide chains. During stress conditions tRNAs can be cleaved to generate tRNA-derived fragments. Several ribonucleases have been identified that cleave tRNA, however mutations in the stress-induced ribonuclease Angiogenin have been identified in a range of neurological disorders including Amyotrophic Lateral Sclerosis, Parkinson’s Disease, and Alzheimer’s Disease, suggesting that tRNA cleavage may be dysregulated in neurological disease. tRNA fragments have been detected in biofluids indicating they may be of use as biomarkers for neurological diseases. There is considerable variability in the methods used to quantify tRFs from size selection, adapter ligation, removal of RNA modifications, and sequence analysis approaches which can make it difficult to reconcile multiple studies. Here we review the biology of transfer RNAs and the biogenesis of tRNA-derived fragments, with a focus on the methods used to identify and quantify tRNA fragments and how different methodological approaches can influence tRNA fragment detection. We provide an overview of current literature on the identification of tRNA fragments in neurological disease models and patient samples, with a focus on circulating tRNA fragments as potential biomarkers of neurological diseases.

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Section: Detection Methodsmentioning

confidence: 97%

Section: Detection Methodsmentioning

confidence: 99%

tRNA fragment biomarkers of Neurological Disease: Challenges and Opportunities

Hogg

2023

MRAJ

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“…Given the known links between tRNA modifications and tsRNAs generation 6,32 , as well as the links between tRNA modifications and oxidative stress responses 7 , we aimed to assess the impact of tRNA modifications in response to various stressors alongside our established stable cell lines (Supp. and downregulation of Queuosine (Q) and its galactosylated form (manQ) (Fig.…”

Section: ➢ Angiogenin Regulates Trna Modificationsmentioning

confidence: 99%

“…It is also important to highlight that the majority of studies investigating the relationship between ANG and tRNA cleavage, whether using genetic approaches to knock-out ANG or by adding recombinant ANG to cells, have primarily focused on tRNA cleavage and tsRNAs generation. However, a significant gap exists as these investigations generally overlook the direct influence of ANG on cell viability in stress response scenarios [30][31][32] . Additionally, the precise connections between ANG and mitochondrial function in stress, particularly in the context of the pathophysiology of diseases like ALS, where ANG is a susceptibility gene 33,34 , are yet to be explored.…”

Section: Introductionmentioning

confidence: 99%

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

Al-Mesitef,

Tominaga,

Mousa

et al. 2024

Preprint

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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.

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“…Indeed, most of the noise in AlkAniline-Seq originates from guanosines, making 8-oxo-guanosine and its downstream ring-opened oxidation products prime suspects. The critical dependence of AlkAniline-Seq on unique 5′-phosphates substantially complicates precise and accurate quantification, since calibration curves between AlkAniline-Seq signals and modification content are not linear for all used scoring systems. ,, Its very low background and exceptional S/N ratio qualify AlkAniline-Seq analyses for full mRNA transcriptomes with a complexity of 10 6 –10 7 nt.…”

Section: Modulating the Rt-signature By Chemical Treatmentmentioning

confidence: 99%

General Principles and Limitations for Detection of RNA Modifications by Sequencing

Motorin,

Helm

2023

Acc. Chem. Res.

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Metrics & MoreArticle Recommendations * sı Supporting Information CONSPECTUS: Among the many analytical methods applied to RNA modifications, a particularly pronounced surge has occurred in the past decade in the field of modification mapping. The occurrence of modifications such as m 6 A in mRNA, albeit known since the 1980s, became amenable to transcriptome-wide analyses through the advent of next-generation sequencing techniques in a rather sudden manner. The term "mapping" here refers to detection of RNA modifications in a sequence context, which has a dramatic impact on the interpretation of biological functions. As a consequence, an impressive number of mapping techniques were published, most in the perspective of what now has become known as "epitranscriptomics". While more and more different modifications were reported to occur in mRNA, conflicting reports and controversial results pointed to a number of technical and theoretical problems rooted in analytics, statistics, and reagents. Rather than finding the proverbial needle in a haystack, the tasks were to determine how many needles of what color in what size of a haystack one was looking at.As the authors of this Account, we think it important to outline the limitations of different mapping methods since many life scientists freshly entering the field confuse the accuracy and precision of modification mapping with that of normal sequencing, which already features numerous caveats by itself. Indeed, we propose here to qualify a specific mapping method by the size of the transcriptome that can be meaningfully analyzed with it. We here focus on high throughput sequencing by Illumina technology, referred to as RNA-Seq. We noted with interest the development of methods for modification detection by other high throughput sequencing platforms that act directly on RNA, e.g., PacBio SMRT and nanopore sequencing, but those are not considered here.In contrast to approaches relying on direct RNA sequencing, current Illumina RNA-Seq protocols require prior conversion of RNA into DNA. This conversion relies on reverse transcription (RT) to create cDNA; thereafter, the cDNA undergoes a sequencing-bysynthesis type of analysis. Thus, a particular behavior of RNA modified nucleotides during the RT-step is a prerequisite for their detection (and quantification) by deep sequencing, and RT properties have great influence on the detection efficiency and reliability. Moreover, the RT-step requires annealing of a synthetic primer, a prerequisite with a crucial impact on library preparation. Thus, all RNA-Seq protocols must feature steps for the introduction of primers, primer landing sites, or adapters on both the RNA 3′-and 5′ends.

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Quantification of substoichiometric modification reveals global tsRNA hypomodification, preferences for angiogenin-mediated tRNA cleavage, and idiosyncratic epitranscriptomes of human neuronal cell-lines

Cited by 10 publications

References 74 publications

tRNA fragment biomarkers of Neurological Disease: Challenges and Opportunities

tRNA fragment biomarkers of Neurological Disease: Challenges and Opportunities

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

General Principles and Limitations for Detection of RNA Modifications by Sequencing

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