scholarly journals HydraPsiSeq: a method for systematic and quantitative mapping of pseudouridines in RNA

2020 ◽  
Vol 48 (19) ◽  
pp. e110-e110 ◽  
Author(s):  
Virginie Marchand ◽  
Florian Pichot ◽  
Paul Neybecker ◽  
Lilia Ayadi ◽  
Valérie Bourguignon-Igel ◽  
...  
Keyword(s):  
Rna Modification ◽  
Clinical Samples ◽  
Mapping Technique ◽  
Rna Modifications ◽  
Quantitative Mapping ◽  
In Vitro Transcripts ◽  
Absolute Measurements ◽  
Generation Sequencing ◽  
Accurate Quantification

Abstract Developing methods for accurate detection of RNA modifications remains a major challenge in epitranscriptomics. Next-generation sequencing-based mapping approaches have recently emerged but, often, they are not quantitative and lack specificity. Pseudouridine (ψ), produced by uridine isomerization, is one of the most abundant RNA modification. ψ mapping classically involves derivatization with soluble carbodiimide (CMCT), which is prone to variation making this approach only semi-quantitative. Here, we developed ‘HydraPsiSeq’, a novel quantitative ψ mapping technique relying on specific protection from hydrazine/aniline cleavage. HydraPsiSeq is quantitative because the obtained signal directly reflects pseudouridine level. Furthermore, normalization to natural unmodified RNA and/or to synthetic in vitro transcripts allows absolute measurements of modification levels. HydraPsiSeq requires minute amounts of RNA (as low as 10–50 ng), making it compatible with high-throughput profiling of diverse biological and clinical samples. Exploring the potential of HydraPsiSeq, we profiled human rRNAs, revealing strong variations in pseudouridylation levels at ∼20–25 positions out of total 104 sites. We also observed the dynamics of rRNA pseudouridylation throughout chondrogenic differentiation of human bone marrow stem cells. In conclusion, HydraPsiSeq is a robust approach for the systematic mapping and accurate quantification of pseudouridines in RNAs with applications in disease, aging, development, differentiation and/or stress response.

scholarly journals Subcellular relocalization and nuclear redistribution of the RNA methyltransferases TRMT1 and TRMT1L upon neuronal activation

2020 ◽  
Author(s):  
Nicky Jonkhout ◽  
Sonia Cruciani ◽  
Helaine Graziele Santos Vieira ◽  
Julia Tran ◽  
Huanle Liu ◽  
...  
Keyword(s):  
Neuronal Plasticity ◽  
Long Term Potentiation ◽  
Neurological Dysfunction ◽  
Rna Modification ◽  
Specific Response ◽  
Environmental Response ◽  
Neuronal Activation ◽  
Rna Modifications ◽  
Environmental Responses

ABSTRACTRNA modifications are dynamic chemical entities that regulate RNA fate, and an avenue for environmental response in neuronal function. However, which RNA modifications may be playing a role in neuronal plasticity and environmental responses is largely unknown. Here we characterize the biochemical function and cellular dynamics of two human RNA methyltransferases previously associated with neurological dysfunction, TRMT1 and its homolog, TRMT1-like (TRMT1L). Using a combination of next-generation sequencing, LC-MS/MS, patient-derived cell lines and knockout mouse models, we confirm the previously reported dimethylguanosine (m 2,2 G) activity of TRMT1 in tRNAs, as well as reveal that TRMT1L, whose activity was unknown, is responsible for methylating a subset of cytosolic tRNA Ala (AGC) isoacceptors at position 26. Using a cellular in vitro model that mimics neuronal activation and long term potentiation, we find that both TRMT1 and TRMT1L change their subcellular localization upon neuronal activation. Specifically, we observe a major subcellular relocalization from mitochondria and other cytoplasmic domains (TRMT1) and nucleoli (TRMT1L) to different small punctate compartments in the nucleus, which are as yet uncharacterized. This phenomenon does not occur upon heat shock, suggesting that the relocalization of TRMT1 and TRMT1L is not a general reaction to stress, but rather a specific response to neuronal activation. Our results suggest that subcellular relocalization of RNA modification enzymes play a role in neuronal plasticity and transmission of information, presumably by addressing new targets.

scholarly journals Rapid and dynamic transcriptome regulation by RNA editing and RNA modifications

2016 ◽  
Vol 213 (1) ◽  
pp. 15-22 ◽  
Author(s):  
Konstantin Licht ◽  
Michael F. Jantsch
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
Rna Editing ◽  
Messenger Rna ◽  
Gene Expression Regulation ◽  
Rna Modification ◽  
Rna Modifications ◽  
Transcriptome Regulation ◽  
Affect Gene Expression ◽  
Generation Sequencing

Advances in next-generation sequencing and mass spectrometry have revealed widespread messenger RNA modifications and RNA editing, with dramatic effects on mammalian transcriptomes. Factors introducing, deleting, or interpreting specific modifications have been identified, and analogous with epigenetic terminology, have been designated “writers,” “erasers,” and “readers.” Such modifications in the transcriptome are referred to as epitranscriptomic changes and represent a fascinating new layer of gene expression regulation that has only recently been appreciated. Here, we outline how RNA editing and RNA modification can rapidly affect gene expression, making both processes as well suited to respond to cellular stress and to regulate the transcriptome during development or circadian periods.

scholarly journals Deep assessment of human disease-associated ribosomal RNA modifications using Nanopore direct RNA sequencing

2021 ◽  
Author(s):  
Isabel S Naarmann-de Vries ◽  
Christiane Zorbas ◽  
Amina Lemsara ◽  
Maja Bencun ◽  
Sarah Schudy ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Clinical Samples ◽  
Sequencing Data ◽  
Rna Modifications ◽  
Flow Cells ◽  
Wide Range ◽  
Catalytically Active ◽  
Induced Pluripotent ◽  
First Time

The catalytically active component of ribosomes, rRNA, is long studied and heavily modified. However, little is known about functional and pathological consequences of changes in human rRNA modification status. Direct RNA sequencing on the Nanopore platform enables the direct assessment of rRNA modifications. We established a targeted Nanopore direct rRNA sequencing approach and applied it to CRISPR-Cas9 engineered HCT116 cells, lacking specific enzymatic activities required to establish defined rRNA base modifications. We analyzed these sequencing data along with wild type samples and in vitro transcribed reference sequences to specifically detect changes in modification status. We show for the first time that direct RNA-sequencing is feasible on smaller, i.e. Flongle, flow cells. Our targeted approach reduces RNA input requirements, making it accessible to the analysis of limited samples such as patient derived material. The analysis of rRNA modifications during cardiomyocyte differentiation of human induced pluripotent stem cells, and of heart biopsies from cardiomyopathy patients revealed altered modifications of specific sites, among them pseudouridine, 2-O-methylation of ribose and acetylation of cytidine. Targeted direct rRNA-seq analysis with JACUSA2 opens up the possibility to analyze dynamic changes in rRNA modifications in a wide range of biological and clinical samples.

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

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.

scholarly journals Long-Reads-Based Metagenomics in Clinical Diagnosis With a Special Focus on Fungal Infections

2022 ◽  
Vol 12 ◽  
Author(s):  
Minh Thuy Vi Hoang ◽  
Laszlo Irinyi ◽  
Yiheng Hu ◽  
Benjamin Schwessinger ◽  
Wieland Meyer
Keyword(s):  
Clinical Diagnosis ◽  
Fungal Infections ◽  
Diagnostic Technique ◽  
Infectious Agent ◽  
Epidemiological Data ◽  
Clinical Samples ◽  
Special Focus ◽  
Long Read ◽  
Generation Sequencing

Identification of the causative infectious agent is essential in the management of infectious diseases, with the ideal diagnostic method being rapid, accurate, and informative, while remaining cost-effective. Traditional diagnostic techniques rely on culturing and cell propagation to isolate and identify the causative pathogen. These techniques are limited by the ability and the time required to grow or propagate an agent in vitro and the facts that identification based on morphological traits are non-specific, insensitive, and reliant on technical expertise. The evolution of next-generation sequencing has revolutionized genomic studies to generate more data at a cheaper cost. These are divided into short- and long-read sequencing technologies, depending on the length of reads generated during sequencing runs. Long-read sequencing also called third-generation sequencing emerged commercially through the instruments released by Pacific Biosciences and Oxford Nanopore Technologies, although relying on different sequencing chemistries, with the first one being more accurate both platforms can generate ultra-long sequence reads. Long-read sequencing is capable of entirely spanning previously established genomic identification regions or potentially small whole genomes, drastically improving the accuracy of the identification of pathogens directly from clinical samples. Long-read sequencing may also provide additional important clinical information, such as antimicrobial resistance profiles and epidemiological data from a single sequencing run. While initial applications of long-read sequencing in clinical diagnosis showed that it could be a promising diagnostic technique, it also has highlighted the need for further optimization. In this review, we show the potential long-read sequencing has in clinical diagnosis of fungal infections and discuss the pros and cons of its implementation.

scholarly journals Nuclear RNA-acetylation can be erased by the deacetylase SIRT7

2021 ◽  
Author(s):  
Pavel Kudrin ◽  
David Meierhofer ◽  
Cathrine Broberg Vågbø ◽  
Ulf Andersson Vang Ørom
Keyword(s):  
Cell Line ◽  
Rna Stability ◽  
Rna Modification ◽  
Rrna Processing ◽  
Rna Modifications ◽  
Oxidative Demethylation ◽  
Deficient Cell ◽  
Ribosomal Rna Processing ◽  
And Function

AbstractA large number of RNA modifications are known to affect processing and function of rRNA, tRNA and mRNA 1. The N4-acetylcytidine (ac4C) is the only known RNA acetylation event and is known to occur on rRNA, tRNA and mRNA 2,3. RNA modification by acetylation affects a number of biological processes, including translation and RNA stability 2. For a few RNA methyl modifications, a reversible nature has been demonstrated where specific writer proteins deposit the modification and eraser proteins can remove them by oxidative demethylation 4–6. The functionality of RNA modifications is often mediated by interaction with reader proteins that bind dependent on the presence of specific modifications 1. The NAT10 acetyltransferase has been firmly identified as the main writer of acetylation of cytidine ribonucleotides, but so far neither readers nor erasers of ac4C have been identified 2,3. Here we show, that ac4C is bound by the nucleolar protein NOP58 and deacetylated by SIRT7, for the first time demonstrating reversal by another mechanism than oxidative demethylation. NOP58 and SIRT7 are involved in snoRNA function and pre-ribosomal RNA processing 7–10, and using a NAT10 deficient cell line we can show that the reduction in ac4C levels affects both snoRNA sub-nuclear localization and pre-rRNA processing. SIRT7 can deacetylate RNA in vitro and endogenous levels of ac4C on snoRNA increase in a SIRT7 deficient cell line, supporting its endogenous function as an RNA deacetylase. In summary, we identify the first eraser and reader proteins of the RNA modification ac4C, respectively, and suggest an involvement of RNA acetylation in snoRNA function and pre-rRNA processing.

The Mechanism of Platelet Aggregation Induced by HLA-Related Antibodies

1996 ◽  
Vol 76 (05) ◽  
pp. 774-779 ◽  
Author(s):  
John T Brandt ◽  
Carmen J Julius ◽  
Jeanne M Osborne ◽  
Clark L Anderson
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Complement Activation ◽  
Thromboembolic Disease ◽  
Clinical Samples ◽  
Hla Antigen ◽  
Aggregation Response ◽  
Immune Mediated ◽  
Dependent Pathway

SummaryImmune-mediated platelet activation is emerging as an important pathogenic mechanism of thrombosis. In vitro studies have suggested two distinct pathways for immune-mediated platelet activation; one involving clustering of platelet FcyRIIa, the other involving platelet-associated complement activation. HLA-related antibodies have been shown to cause platelet aggregation, but the mechanism has not been clarified. We evaluated the mechanism of platelet aggregation induced by HLA-related antibodies from nine patients. Antibody to platelet FcyRIIa failed to block platelet aggregation with 8/9 samples, indicating that engagement of platelet FcyRIIa is not necessary for the platelet aggregation induced by HLA-related antibodies. In contrast, platelet aggregation was blocked by antibodies to human C8 (5/7) or C9 (7/7). F(ab’)2 fragments of patient IgG failed to induce platelet activation although they bound to HLA antigen on platelets. Intact patient IgG failed to aggregate washed platelets unless aged serum was added. The activating IgG could be adsorbed by incubation with lymphocytes and eluted from the lymphocytes. These results indicate that complement activation is involved in the aggregation response to HLA-related antibodies. This is the first demonstration of complement-mediated platelet aggregation by clinical samples. Five of the patients developed thrombocytopenia in relationship to blood transfusion and two patients developed acute thromboembolic disease, suggesting that these antibodies and the complement-dependent pathway of platelet aggregation may be of clinical significance.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Efficient Peptide-Mediated In Vitro Delivery of Cas9 RNP

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 878
Author(s):  
Oskar Gustafsson ◽  
Julia Rädler ◽  
Samantha Roudi ◽  
Tõnis Lehto ◽  
Mattias Hällbrink ◽  
...  
Keyword(s):  
Freeze Drying ◽  
Cell Penetrating Peptide ◽  
Wild Type ◽  
Efficient Manner ◽  
Editing Efficiency ◽  
Freeze Thaw ◽  
Enzyme Digest ◽  
Clinical Potential ◽  
Generation Sequencing

The toolbox for genetic engineering has quickly evolved from CRISPR/Cas9 to a myriad of different gene editors, each with promising properties and enormous clinical potential. However, a major challenge remains: delivering the CRISPR machinery to the nucleus of recipient cells in a nontoxic and efficient manner. In this article, we repurpose an RNA-delivering cell-penetrating peptide, PepFect14 (PF14), to deliver Cas9 ribonucleoprotein (RNP). The RNP-CPP complex achieved high editing rates, e.g., up to 80% in HEK293T cells, while being active at low nanomolar ranges without any apparent signs of toxicity. The editing efficiency was similar to or better compared to the commercially available reagents RNAiMAX and CRISPRMax. The efficiency was thoroughly evaluated in reporter cells and wild-type cells by restriction enzyme digest and next-generation sequencing. Furthermore, the CPP-Cas9-RNP complexes were demonstrated to withstand storage at different conditions, including freeze-thaw cycles and freeze-drying, without a loss in editing efficiency. This CPP-based delivery strategy complements existing technologies and further opens up new opportunities for Cas9 RNP delivery, which can likely be extended to other gene editors in the future.

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