scholarly journals The Stress-Dependent Dynamics of Saccharomyces cerevisiae tRNA and rRNA Modification Profiles

Genes ◽  
2021 ◽  
Vol 12 (9) ◽  
pp. 1344
Author(s):  
Yasemin Yoluç ◽  
Erik van de Logt ◽  
Stefanie Kellner-Kaiser
Keyword(s):  
Isotope Labeling ◽  
Model Organism ◽  
Rna Degradation ◽  
Rna Modification ◽  
Rna Modifications ◽  
Profile Changes ◽  
Stressed Cells ◽  
As Stress ◽  
Stress Dependent ◽  
Internal And External Factors

RNAs are key players in the cell, and to fulfil their functions, they are enzymatically modified. These modifications have been found to be dynamic and dependent on internal and external factors, such as stress. In this study we used nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) to address the question of which mechanisms allow the dynamic adaptation of RNA modifications during stress in the model organism S. cerevisiae. We found that both tRNA and rRNA transcription is stalled in yeast exposed to stressors such as H2O2, NaAsO2 or methyl methanesulfonate (MMS). From the absence of new transcripts, we concluded that most RNA modification profile changes observed to date are linked to changes happening on the pre-existing RNAs. We confirmed these changes, and we followed the fate of the pre-existing tRNAs and rRNAs during stress recovery. For MMS, we found previously described damage products in tRNA, and in addition, we found evidence for direct base methylation damage of 2′O-ribose methylated nucleosides in rRNA. While we found no evidence for increased RNA degradation after MMS exposure, we observed rapid loss of all methylation damages in all studied RNAs. With NAIL-MS we further established the modification speed in new tRNA and 18S and 25S rRNA from unstressed S. cerevisiae. During stress exposure, the placement of modifications was delayed overall. Only the tRNA modifications 1-methyladenosine and pseudouridine were incorporated as fast in stressed cells as in control cells. Similarly, 2′-O-methyladenosine in both 18S and 25S rRNA was unaffected by the stressor, but all other rRNA modifications were incorporated after a delay. In summary, we present mechanistic insights into stress-dependent RNA modification profiling in S. cerevisiae tRNA and rRNA.

scholarly journals Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis

Genes ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 26 ◽  
Author(s):  
Kayla Borland ◽  
Jan Diesend ◽  
Taku Ito-Kureha ◽  
Vigo Heissmeyer ◽  
Christian Hammann ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Isotope Labeling ◽  
Rna Stability ◽  
Internal Standard ◽  
Modified Nucleosides ◽  
Rna Modification ◽  
Mouse Tissue ◽  
Rna Modifications ◽  
Internal Standards ◽  
Translation Fidelity

Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue.

Hypoxia-induced alteration of RNA modifications in the mouse testis and sperm

2021 ◽  
Author(s):  
Tong He ◽  
Huanping Guo ◽  
Xipeng Shen ◽  
Xiao Wu ◽  
Lin Xia ◽  
...  
Keyword(s):  
Hypobaric Hypoxia ◽  
Environmental Changes ◽  
Epigenetic Inheritance ◽  
Transcriptional Regulators ◽  
Extreme Environment ◽  
Mouse Testis ◽  
Rna Modification ◽  
Rna Modifications ◽  
Sperm Counts ◽  
Health Studies

Abstract Hypobaric hypoxia as an extreme environment in a plateau may have deleterious effects on human health. Studies have indicated that rush entry into a plateau may reduce male fertility and manifest in decreased sperm counts and weakened sperm motility. RNA modifications are sensitive to environmental changes and have recently emerged as novel post-transcriptional regulators in male spermatogenesis and intergenerational epigenetic inheritance. In the present study, we generated a mouse hypoxia model simulating the environment of 5500 meters in altitude for 35 days, which led to compromised spermatogenesis, decreased sperm counts, and an increased sperm deformation rate. Using this hypoxia model, we further applied our recently developed high-throughput RNA modification quantification platform based on LC–MS/MS, which exhibited the capacity to simultaneously examine 25 types of RNA modifications. Our results revealed an altered sperm RNA modifications signature in the testis (6 types) and mature sperm (11 types) under the hypoxia model, with 4 types showing overlap (Am, Gm, m7G, and m22G). Our data first drew the signature of RNA modification profiles and comprehensively analyzed the alteration of RNA modification levels in mouse testis and sperm under a mouse hypoxia model. These data may be highly related to human conditions under a similar hypoxia environment.

scholarly journals RNA Modification Level Estimation with pulseR

Genes ◽  
2018 ◽  
Vol 9 (12) ◽  
pp. 619
Author(s):  
Etienne Boileau ◽  
Christoph Dieterich
Keyword(s):  
Experimental Approach ◽  
High Efficiency ◽  
Rna Modification ◽  
Model Parameters ◽  
Metabolic Labeling ◽  
Rna Seq ◽  
Rna Modifications ◽  
Log Odds ◽  
Pros And Cons ◽  
Wide Scale

RNA modifications regulate the complex life of transcripts. An experimental approach called LAIC-seq was developed to characterize modification levels on a transcriptome-wide scale. In this method, the modified and unmodified molecules are separated using antibodies specific for a given RNA modification (e.g., m6A). In essence, the procedure of biochemical separation yields three fractions: Input, eluate, and supernatent, which are subjected to RNA-seq. In this work, we present a bioinformatics workflow, which starts from RNA-seq data to infer gene-specific modification levels by a statistical model on a transcriptome-wide scale. Our workflow centers around the pulseR package, which was originally developed for the analysis of metabolic labeling experiments. We demonstrate how to analyze data without external normalization (i.e., in the absence of spike-ins), given high efficiency of separation, and how, alternatively, scaling factors can be derived from unmodified spike-ins. Importantly, our workflow provides an estimate of uncertainty of modification levels in terms of confidence intervals for model parameters, such as gene expression and RNA modification levels. We also compare alternative model parametrizations, log-odds, or the proportion of the modified molecules and discuss the pros and cons of each representation. In summary, our workflow is a versatile approach to RNA modification level estimation, which is open to any read-count-based experimental approach.

Trivariant Lagrange Factor Polynomial for Discrete Rayleigh Distributed Reliability Analysis of Aeroengine Combustion Chamber Cylinder

2013 ◽  
Vol 10 (1) ◽  
Author(s):  
Chun Nam Wong ◽  
Yang Lu
Keyword(s):  
Combustion Chamber ◽  
Thermal Conditions ◽  
Economic Factors ◽  
Structural Variants ◽  
Temperature Dependent ◽  
Application Range ◽  
Linear Polynomial ◽  
As Stress ◽  
Stress Dependent ◽  
Dependent Probability

In most of the existing stress-strength interference (SSI) models, stress and strength are assumed to be independent structural variants. However, under severe thermal conditions, such as in aeroengine combustion chamber, this assumption may not hold. One structural variant, such as strength, may become unilateral dependent on another variant, such as stress or temperature. In addition, to evaluate the discrete reliability of structures using unilateral dependent structural variants, discrete SSI models were developed using not just linear polynomial or line segments, but higher order polynomials. These models are based on the trivariant Lagrange factor polynomial approach. Normal distributed temperature dependent stress and Rayleigh distributed thermal stress dependent strength are represented by discrete structural variants that possess unilateral dependent probability mean functions. Based on their dependence formulations, the trivariant Lagrange factor polynomial of the discrete SSI model was generated. Applicability of this method was validated by a specific aeroengine combustion chamber cylinder using different molding alloys. Meanwhile the application range of some existing SSI models is extended for interval shifted data. Comparing machinability, reliability, and economic factors, 1Cr11MoV was the most suitable alloy in the design.

scholarly journals The architecture of SARS-CoV-2 transcriptome

2020 ◽  
Author(s):  
Dongwan Kim ◽  
Joo-Yeon Lee ◽  
Jeong-Sun Yang ◽  
Jun Won Kim ◽  
V. Narry Kim ◽  
...  
Keyword(s):  
High Resolution ◽  
Rna Sequencing ◽  
Rna Modification ◽  
List Type ◽  
Rna Modifications ◽  
Subgenomic Rnas ◽  
Frequent Motif ◽  
High Resolution Map ◽  
Functional Investigation ◽  
Resolution Map

AbstractSARS-CoV-2 is a betacoronavirus that is responsible for the COVID-19 pandemic. The genome of SARS-CoV-2 was reported recently, but its transcriptomic architecture is unknown. Utilizing two complementary sequencing techniques, we here present a high-resolution map of the SARS-CoV-2 transcriptome and epitranscriptome. DNA nanoball sequencing shows that the transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical. In addition to the genomic RNA and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces a large number of transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Using nanopore direct RNA sequencing, we further find at least 41 RNA modification sites on viral transcripts, with the most frequent motif being AAGAA. Modified RNAs have shorter poly(A) tails than unmodified RNAs, suggesting a link between the internal modification and the 3′ tail. Functional investigation of the unknown ORFs and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2.HighlightsWe provide a high-resolution map of SARS-CoV-2 transcriptome and epitranscriptome using nanopore direct RNA sequencing and DNA nanoball sequencing.The transcriptome is highly complex owing to numerous recombination events, both canonical and noncanonical.In addition to the genomic and subgenomic RNAs common in all coronaviruses, SARS-CoV-2 produces transcripts encoding unknown ORFs.We discover at least 41 potential RNA modification sites with an AAGAA motif.

RNA modifications in hematopoietic malignancies: A new research frontier

Blood ◽  
2021 ◽  
Author(s):  
Ying Qing ◽  
Rui Su ◽  
Jianjun Chen
Keyword(s):  
Myeloid Leukemia ◽  
Therapeutic Potential ◽  
Rna Modification ◽  
Biological Functions ◽  
Rna Modifications ◽  
Aberrant Expression ◽  
Protein Coding ◽  
Hematopoietic Malignancies ◽  
Underlying Mechanisms ◽  
New Research

Both protein-coding and noncoding RNAs can be decorated with a wealth of chemical modifications and such modifications coordinately orchestrate gene expression during normal hematopoietic differentiation and development. However, aberrant expression and/or dysfunction of the relevant RNA modification modulators/regulators ("writers", "erasers", and "readers") drive the initiation and progression of hematopoietic malignancies, and targeting these dysregulated modulators holds potent therapeutic potential for the treatment of hematopoietic malignancies. In this review, we summarize current progress in the understanding of the biological functions and underlying mechanisms of RNA modifications in normal and malignant hematopoiesis, with a focus on the N6-methyladenosine (m6A) modification, and discuss the therapeutic potential of targeting RNA modifications for the treatment of hematopoietic malignancies, especially acute myeloid leukemia (AML).

scholarly journals Nucleotide resolution profiling of m3C RNA modification by HAC-seq

2020 ◽  
Author(s):  
Jia Cui ◽  
Qi Liu ◽  
Erdem Sendinc ◽  
Yang Shi ◽  
Richard I Gregory
Keyword(s):  
Rna Modification ◽  
Rna Modifications ◽  
Mcf7 Cells ◽  
Single Nucleotide ◽  
Novel Method ◽  
Specific Mapping ◽  
Nucleotide Resolution ◽  
And Function ◽  
Trna Isoacceptors ◽  
Single Nucleotide Resolution

Abstract Cellular RNAs are subject to a myriad of different chemical modifications that play important roles in controlling RNA expression and function. Dysregulation of certain RNA modifications, the so-called ‘epitranscriptome’, contributes to human disease. One limitation in studying the functional, physiological, and pathological roles of the epitranscriptome is the availability of methods for the precise mapping of individual RNA modifications throughout the transcriptome. 3-Methylcytidine (m3C) modification of certain tRNAs is well established and was also recently detected in mRNA. However, methods for the specific mapping of m3C throughout the transcriptome are lacking. Here, we developed a m3C-specific technique, Hydrazine-Aniline Cleavage sequencing (HAC-seq), to profile the m3C methylome at single-nucleotide resolution. We applied HAC-seq to analyze ribosomal RNA (rRNA)-depleted total RNAs in human cells. We found that tRNAs are the predominant m3C-modified RNA species, with 17 m3C modification sites on 11 cytoplasmic and 2 mitochondrial tRNA isoacceptors in MCF7 cells. We found no evidence for m3C-modification of mRNA or other non-coding RNAs at comparable levels to tRNAs in these cells. HAC-seq provides a novel method for the unbiased, transcriptome-wide identification of m3C RNA modification at single-nucleotide resolution, and could be widely applied to reveal the m3C methylome in different cells and tissues.

Evaluate the Importance of Shear Modulus in Dynamic FEM of Flexible Pavement Considering AC Cross-Anisotropy

2016 ◽  
Author(s):  
Mesbah U. Ahmed ◽  
Rafiqul A. Tarefder
Keyword(s):  
Shear Modulus ◽  
Resilient Modulus ◽  
Vertical Plane ◽  
Element Model ◽  
Tire Pressure ◽  
Stress Strain ◽  
Viscoelastic Parameters ◽  
Cross Anisotropy ◽  
As Stress ◽  
Stress Dependent

Goal of this study is to evaluate the effect of shear modulus variation on pavement responses, such as stress-strain, under dynamic load incorporating the AC cross-anisotropy. A dynamic Finite Element Model (FEM) of an instrumented asphalt pavement section on Interstate 40 (I-40) near Albuquerque, New Mexico, is developed in ABAQUS to determine stress-strain under truck tire pressure. Laboratory dynamic modulus tests were conducted on the AC cores to determine the temperature and frequency varying modulus values along both vertical and horizontal directions. The test outcomes are used to produce cross-anisotropic and viscoelastic parameters. Resilient modulus tests are conducted on granular aggregates from base and subbase layer to determine the nonlinear elastic and stress-dependent modulus values. These material parameters are integrated to the FEM through a FORTRAN subroutine via User Defined Material (UMAT) in the ABAQUS. The developed FEM is validated using the pavement deflections and stress-strain data under Falling Weight Deflectometer (FWD) test. The validated dynamic FEM is simulated under the non-uniform vertical tire contact stress. For the parametric study to investigate the effect of shear modulus variation on pavement responses, the validated FEM is simulated by varying the shear modulus in the AC layer. The results show that the variation in shear modulus along a vertical plane barely affects the tensile strain at the bottom of the AC layer and vertical compressive strains in both AC and unbound layers.

scholarly journals Leaf Senescence: The Chloroplast Connection Comes of Age

Plants ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 495 ◽  
Author(s):  
Martín L. Mayta ◽  
Mohammad-Reza Hajirezaei ◽  
Néstor Carrillo ◽  
Anabella F. Lodeyro
Keyword(s):  
Cell Death ◽  
Leaf Senescence ◽  
Developmental Process ◽  
Reproductive Organs ◽  
Common Thread ◽  
Dependent Cell ◽  
Redox Poise ◽  
Natural Leaf ◽  
As Stress ◽  
Stress Dependent

Leaf senescence is a developmental process critical for plant fitness, which involves genetically controlled cell death and ordered disassembly of macromolecules for reallocating nutrients to juvenile and reproductive organs. While natural leaf senescence is primarily associated with aging, it can also be induced by environmental and nutritional inputs including biotic and abiotic stresses, darkness, phytohormones and oxidants. Reactive oxygen species (ROS) are a common thread in stress-dependent cell death and also increase during leaf senescence. Involvement of chloroplast redox chemistry (including ROS propagation) in modulating cell death is well supported, with photosynthesis playing a crucial role in providing redox-based signals to this process. While chloroplast contribution to senescence received less attention, recent findings indicate that changes in the redox poise of these organelles strongly affect senescence timing and progress. In this review, the involvement of chloroplasts in leaf senescence execution is critically assessed in relation to available evidence and the role played by environmental and developmental cues such as stress and phytohormones. The collected results indicate that chloroplasts could cooperate with other redox sources (e.g., mitochondria) and signaling molecules to initiate the committed steps of leaf senescence for a best use of the recycled nutrients in plant reproduction.

scholarly journals Human RNA cap1 methyltransferase CMTr1 cooperates with RNA helicase DHX15 to modify RNAs with highly structured 5′ termini

2018 ◽  
Vol 373 (1762) ◽  
pp. 20180161 ◽  
Author(s):  
Diana Toczydlowska-Socha ◽  
Magdalena M. Zielinska ◽  
Malgorzata Kurkowska ◽  
Astha ◽  
Catarina F. Almeida ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Degradation ◽  
Rna Modification ◽  
Structure Characteristic ◽  
Helicase Activity ◽  
Theme Issue ◽  
Rna Capping ◽  
Advantageous Effect

The 5′-cap structure, characteristic for RNA polymerase II-transcribed RNAs, plays important roles in RNA metabolism. In humans, RNA cap formation includes post-transcriptional modification of the first transcribed nucleotide by RNA cap1 methyltransferase (CMTr1). Here, we report that CMTr1 activity is hindered towards RNA substrates with highly structured 5′ termini. We found that CMTr1 binds ATP-dependent RNA DHX15 helicase and that this interaction, mediated by the G-patch domain of CMTr1, has an advantageous effect on CMTr1 activity towards highly structured RNA substrates. The effect of DHX15 helicase activity is consistent with the strength of the secondary structure that has to be removed for CMTr1 to access the 5′-terminal residues in a single-stranded conformation. This is, to our knowledge, the first demonstration of the involvement of DHX15 in post-transcriptional RNA modification, and the first example of a molecular process in which DHX15 directly affects the activity of another enzyme. Our findings suggest a new mechanism underlying the regulatory role of DHX15 in the RNA capping process. RNAs with highly structured 5′ termini constitute a significant fraction of the human transcriptome. Hence, CMTr1–DHX15 cooperation is likely to be important for the metabolism of RNA polymerase II-transcribed RNAs. This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.

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