A dual-purpose polymerase engineered for direct sequencing of pseudouridine and queuosine

Huber, Luisa B; Kaur, Navpreet; Henkel, Melanie; Marchand, Virginie; Motorin, Yuri; Ehrenhofer‐Murray, Ann E.; Marx, Andreas

doi:10.1093/nar/gkad177

Cited by 10 publications

(5 citation statements)

References 60 publications

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“…The development of new reagents and methods for mapping modifications by high-throughput sequencing has received particular attention here, especially in the early phase of the program. Here, the focus has been visible in numerous high-level publications describing, e.g., cofactors for modification enzymes, special polymerases and chemical derivatizations, − as well as catalytic nucleic acids , for computer-assisted mapping of RNA modifications in a sequence context . Another increasingly noted problem is the visualization of modifications, e.g., in a cell …”

Section: Results: the Three Guiding Questions: Where How And Why?mentioning

confidence: 99%

Experience with German Research Consortia in the Field of Chemical Biology of Native Nucleic Acid Modifications

Helm,

Bohnsack,

Carell

et al. 2023

ACS Chem. Biol.

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The chemical biology of native nucleic acid modifications has seen an intense upswing, first concerning DNA modifications in the field of epigenetics and then concerning RNA modifications in a field that was correspondingly rebaptized epitranscriptomics by analogy. The German Research Foundation (DFG) has funded several consortia with a scientific focus in these fields, strengthening the traditionally well-developed nucleic acid chemistry community and inciting it to team up with colleagues from the life sciences and data science to tackle interdisciplinary challenges. This Perspective focuses on the genesis, scientific outcome, and downstream impact of the DFG priority program SPP1784 and offers insight into how it fecundated further consortia in the field. Pertinent research was funded from mid-2015 to 2022, including an extension related to the coronavirus pandemic. Despite being a detriment to research activity in general, the pandemic has resulted in tremendously boosted interest in the field of RNA and RNA modifications as a consequence of their widespread and successful use in vaccination campaigns against SARS-CoV-2. Funded principal investigators published over 250 pertinent papers with a very substantial impact on the field. The program also helped to redirect numerous laboratories toward this dynamic field. Finally, SPP1784 spawned initiatives for several funded consortia that continue to drive the fields of nucleic acid modification.

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Section: Results: the Three Guiding Questions: Where How And Why?mentioning

confidence: 99%

Experience with German Research Consortia in the Field of Chemical Biology of Native Nucleic Acid Modifications

Helm,

Bohnsack,

Carell

et al. 2023

ACS Chem. Biol.

Self Cite

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“…We applied this to tRNAs obtained both by in vitro transcription as well as ex cellulo from S. pombe and E. coli . This approach allows the detection of these modifications at single-base resolution, and it overcomes limitations caused by other methods for Q detection, such as prior chemical treatment or an error-prone polymerase ( 46 , 48 ).…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, in our earlier work, we labeled tRNAs in vitro and in vivo with a non-natural preQ 1 derivative carrying an azide group, which, when combined with RNA-seq, can reveal the native substrates of TGT in vivo ( 47 ). Also, we recently developed a specialized reverse transcriptase/ polymerase that makes misincorporations opposite queuosine and pseudouridine and thus can be employed to detect Q modification by analyzing error profiles after NGS ( 48 ). However, as with earlier NGS-based approaches, these methods require chemical treatment and/or reverse transcription, which have the aforementioned disadvantages.…”

Section: Introductionmentioning

confidence: 99%

Detection of queuosine and queuosine precursors in tRNAs by direct RNA sequencing

Sun,

Piechotta,

Naarmann-de Vries

et al. 2023

Nucleic Acids Research

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Queuosine (Q) is a complex tRNA modification found in bacteria and eukaryotes at position 34 of four tRNAs with a GUN anticodon, and it regulates the translational efficiency and fidelity of the respective codons that differ at the Wobble position. In bacteria, the biosynthesis of Q involves two precursors, preQ0 and preQ1, whereas eukaryotes directly obtain Q from bacterial sources. The study of queuosine has been challenging due to the limited availability of high-throughput methods for its detection and analysis. Here, we have employed direct RNA sequencing using nanopore technology to detect the modification of tRNAs with Q and Q precursors. These modifications were detected with high accuracy on synthetic tRNAs as well as on tRNAs extracted from Schizosaccharomyces pombe and Escherichia coli by comparing unmodified to modified tRNAs using the tool JACUSA2. Furthermore, we present an improved protocol for the alignment of raw sequence reads that gives high specificity and recall for tRNAs ex cellulo that, by nature, carry multiple modifications. Altogether, our results show that 7-deazaguanine-derivatives such as queuosine are readily detectable using direct RNA sequencing. This advancement opens up new possibilities for investigating these modifications in native tRNAs, furthering our understanding of their biological function.

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“…The development of the reverse transcriptase-active DNA polymerase variant RT-KTq I614Y marks a significant advancement in RNA modification research. When paired with next-generation sequencing, this technology allows direct identification of pseudouridine and queuosine (Q) modifications in RNA without prior treatment, providing new insights into the effects of pseudouridylation on cellular mechanisms [112]. Yeast strains lacking Pus3 show increased protein aggregation and activation of stress response pathways, highlighting the enzyme's crucial role [113,114].…”

Section: Pseudouridylation (ψ)mentioning

confidence: 99%

tRNA Modifications and Dysregulation: Implications for Brain Diseases

Lv,

Zhang,

et al. 2024

Brain Sciences

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Transfer RNAs (tRNAs) are well-known for their essential function in protein synthesis. Recent research has revealed a diverse range of chemical modifications that tRNAs undergo, which are crucial for various cellular processes. These modifications are necessary for the precise and efficient translation of proteins and also play important roles in gene expression regulation and cellular stress response. This review examines the role of tRNA modifications and dysregulation in the pathophysiology of various brain diseases, including epilepsy, stroke, neurodevelopmental disorders, brain tumors, Alzheimer’s disease, and Parkinson’s disease. Through a comprehensive analysis of existing research, our study aims to elucidate the intricate relationship between tRNA dysregulation and brain diseases. This underscores the critical need for ongoing exploration in this field and provides valuable insights that could facilitate the development of innovative diagnostic tools and therapeutic approaches, ultimately improving outcomes for individuals grappling with complex neurological conditions.

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A dual-purpose polymerase engineered for direct sequencing of pseudouridine and queuosine

Cited by 10 publications

References 60 publications

Experience with German Research Consortia in the Field of Chemical Biology of Native Nucleic Acid Modifications

Experience with German Research Consortia in the Field of Chemical Biology of Native Nucleic Acid Modifications

Detection of queuosine and queuosine precursors in tRNAs by direct RNA sequencing

tRNA Modifications and Dysregulation: Implications for Brain Diseases

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