scholarly journals Programmable System of Cas13-Mediated RNA Modification and Its Biological and Biomedical Applications

2021 ◽  
Vol 9 ◽  
Author(s):  
Tian Tang ◽  
Yingli Han ◽  
Yuran Wang ◽  
He Huang ◽  
Pengxu Qian
Keyword(s):  
Cell Fate ◽  
Rna Editing ◽  
Cell Biology ◽  
Rna Stability ◽  
Rna Modification ◽  
Translation Efficiency ◽  
Rna Modifications ◽  
History Of ◽  
Rna Interaction ◽  
Adenosine Deamination

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13 has drawn broad interest to control gene expression and cell fate at the RNA level in general. Apart from RNA interference mediated by its endonuclease activity, the nuclease-deactivated form of Cas13 further provides a versatile RNA-guided RNA-targeting platform for manipulating kinds of RNA modifications post-transcriptionally. Chemical modifications modulate various aspects of RNA fate, including translation efficiency, alternative splicing, RNA–protein affinity, RNA–RNA interaction, RNA stability and RNA translocation, which ultimately orchestrate cellular biologic activities. This review summarizes the history of the CRISPR-Cas13 system, fundamental components of RNA modifications and the related physiological and pathological functions. We focus on the development of epi-transcriptional editing toolkits based on catalytically inactive Cas13, including RNA Editing for Programmable A to I Replacement (REPAIR) and xABE (adenosine base editor) for adenosine deamination, RNA Editing for Specific C-to-U Exchange (RESCUE) and xCBE (cytidine base editor) for cytidine deamination and dm6ACRISPR, as well as the targeted RNA methylation (TRM) and photoactivatable RNA m6A editing system using CRISPR-dCas13 (PAMEC) for m6A editing. We further highlight the emerging applications of these useful toolkits in cell biology, disease and imaging. Finally, we discuss the potential limitations, such as off-target editing, low editing efficiency and limitation for AAV delivery, and provide possible optimization strategies.

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.

scholarly journals 5-Methylcytosine RNA Modifications Promote Retrovirus Replication in an ALYREF Reader Protein-Dependent Manner

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Matthew Eckwahl ◽  
Ruyi Xu ◽  
Julia Michalkiewicz ◽  
Wen Zhang ◽  
Pooja Patel ◽  
...  
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rna Stability ◽  
Translation Efficiency ◽  
Dependent Manner ◽  
Rna Modifications ◽  
Nucleotide Resolution ◽  
And Function ◽  
Murine Leukemia ◽  
Retrovirus Replication

ABSTRACT RNA modifications play diverse roles in regulating RNA function, and viruses co-opt these pathways for their own benefit. While recent studies have highlighted the importance of N6-methyladenosine (m6A)—the most abundant mRNA modification—in regulating retrovirus replication, the identification and function of other RNA modifications in viral biology have been largely unexplored. Here, we characterized the RNA modifications present in a model retrovirus, murine leukemia virus (MLV), using mass spectrometry and sequencing. We found that 5-methylcytosine (m5C) is highly enriched in viral genomic RNA relative to uninfected cellular mRNAs, and we mapped at single-nucleotide resolution the m5C sites, which are located in multiple clusters throughout the MLV genome. Further, we showed that the m5C reader protein ALYREF plays an important role in regulating MLV replication. Together, our results provide a complete m5C profile in a virus and its function in a eukaryotic mRNA. IMPORTANCE Over 130 modifications have been identified in cellular RNAs, which play critical roles in many cellular processes, from modulating RNA stability to altering translation efficiency. One such modification, 5-methylcytosine, is relatively abundant in mammalian mRNAs, but its precise location and function are not well understood. In this study, we identified unexpectedly high levels of m5C in the murine leukemia virus RNA, precisely mapped its location, and showed that ALYREF, a reader protein that specifically recognizes m5C, regulates viral production. Together, our findings provide a high-resolution atlas of m5C in murine leukemia virus and reveal a functional role of m5C in viral replication.

scholarly journals Single-nucleotide variants in human RNA: RNA editing and beyond

2018 ◽  
Vol 18 (1) ◽  
pp. 30-39 ◽  
Author(s):  
Yan Guo ◽  
Hui Yu ◽  
David C Samuels ◽  
Wei Yue ◽  
Scott Ness ◽  
...  
Keyword(s):  
Rna Editing ◽  
Rna Seq ◽  
Rna Modifications ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Genomic Variants ◽  
High Prevalence ◽  
History Of ◽  
Gene Expression Quantification

Abstract Through analysis of paired high-throughput DNA-Seq and RNA-Seq data, researchers quickly recognized that RNA-Seq can be used for more than just gene expression quantification. The alternative applications of RNA-Seq data are abundant, and we are particularly interested in its usefulness for detecting single-nucleotide variants, which arise from RNA editing, genomic variants and other RNA modifications. A stunning discovery made from RNA-Seq analyses is the unexpectedly high prevalence of RNA-editing events, many of which cannot be explained by known RNA-editing mechanisms. Over the past 6–7 years, substantial efforts have been made to maximize the potential of RNA-Seq data. In this review we describe the controversial history of mining RNA-editing events from RNA-Seq data and the corresponding development of methodologies to identify, predict, assess the quality of and catalog RNA-editing events as well as genomic variants.

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 RNA methylation in mammalian development and cancer

2021 ◽  
Author(s):  
Peizhe Song ◽  
Subiding Tayier ◽  
Zhihe Cai ◽  
Guifang Jia
Keyword(s):  
Cell Fate ◽  
Cancer Progression ◽  
Rna Modification ◽  
Biological Processes ◽  
Cell Fate Determination ◽  
Mammalian Development ◽  
Rna Modifications ◽  
Temporal And Spatial ◽  
Underlying Mechanisms ◽  
Cellular Components

AbstractSimilar to epigenetic DNA and histone modifications, epitranscriptomic modifications (RNA modifications) have emerged as crucial regulators in temporal and spatial gene expression during eukaryotic development. To date, over 170 diverse types of chemical modifications have been identified upon RNA nucleobases. Some of these post-synthesized modifications can be reversibly installed, removed, and decoded by their specific cellular components and play critical roles in different biological processes. Accordingly, dysregulation of RNA modification effectors is tightly orchestrated with developmental processes. Here, we particularly focus on three well-studied RNA modifications, including N6-methyladenosine (m6A), 5-methylcytosine (m5C), and N1-methyladenosine (m1A), and summarize recent knowledge of underlying mechanisms and critical roles of these RNA modifications in stem cell fate determination, embryonic development, and cancer progression, providing a better understanding of the whole association between epitranscriptomic regulation and mammalian development.

scholarly journals Deep and accurate detection of m6A RNA modifications using miCLIP2 and m6Aboost machine learning

2020 ◽  
Author(s):  
Nadine Körtel ◽  
Cornelia Rücklé ◽  
You Zhou ◽  
Anke Busch ◽  
FX Reymond Sutandy ◽  
...  
Keyword(s):  
Machine Learning ◽  
Rna Stability ◽  
Rna Modification ◽  
List Type ◽  
Rna Modifications ◽  
Uv Crosslinking ◽  
Input Material ◽  
Machine Learning Model ◽  
Eukaryotic Mrnas ◽  
Nucleotide Resolution

AbstractN6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic mRNAs and influences many aspects of RNA processing, such as RNA stability and translation. miCLIP (m6A individual-nucleotide resolution UV crosslinking and immunoprecipitation) is an antibody-based approach to map m6A sites in the transcriptome with single-nucleotide resolution. However, due to broad antibody reactivity, reliable identification of m6A sites from miCLIP data remains challenging. Here, we present several experimental and computational innovations, that significantly improve transcriptome-wide detection of m6A sites. Based on the recently developed iCLIP2 protocol, the optimised miCLIP2 results in high-complexity libraries from less input material, which yields a more comprehensive representation of m6A sites. Next, we established a robust computational pipeline to identify true m6A sites from our miCLIP2 data. The analyses are calibrated with data from Mettl3 knockout cells to learn the characteristics of m6A deposition, including a significant number of m6A sites outside of DRACH motifs. In order to make these results universally applicable, we trained a machine learning model, m6Aboost, based on the experimental and RNA sequence features. Importantly, m6Aboost allows prediction of genuine m6A sites in miCLIP data without filtering for DRACH motifs or the need for Mettl3 depletion. Using m6Aboost, we identify thousands of high-confidence m6A sites in different murine and human cell lines, which provide a rich resource for future analysis. Collectively, our combined experimental and computational methodology greatly improves m6A identification.HighlightsmiCLIP2 produces complex libraries to map m6A RNA modificationsMettl3 KO miCLIP2 allows to identify Mettl3-dependent RNA modification sitesMachine learning predicts genuine m6A sites from human and mouse miCLIP2 data without Mettl3 KOm6A modifications frequently occur outside of DRACH motifs and associates with alternative splicing

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.

scholarly journals Regulation of RNA stability at the 3′ end

2020 ◽  
Vol 0 (0) ◽  
Author(s):  
Mallory I. Frederick ◽  
Ilka U. Heinemann
Keyword(s):  
Mrna Decay ◽  
Rna Stability ◽  
Rna Modifications ◽  
Dual Roles ◽  
Cellular Pathways ◽  
Low Levels

AbstractRNA homeostasis is regulated by a multitude of cellular pathways. Although the addition of untemplated adenine residues to the 3′ end of mRNAs has long been known to affect RNA stability, newly developed techniques for 3′-end sequencing of RNAs have revealed various unexpected RNA modifications. Among these, uridylation is most recognized for its role in mRNA decay but is also a key regulator of numerous RNA species, including miRNAs and tRNAs, with dual roles in both stability and maturation of miRNAs. Additionally, low levels of untemplated guanidine and cytidine residues have been observed as parts of more complex tailing patterns.

scholarly journals Metabolic Control of m6A RNA Modification

Metabolites ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 80
Author(s):  
Joohwan Kim ◽  
Gina Lee
Keyword(s):  
Cell Fate ◽  
Metabolic Pathways ◽  
Epigenetic Modification ◽  
Genetic Material ◽  
Rna Modification ◽  
Cell Fate Decisions ◽  
Disease States ◽  
Evolutionarily Conserved ◽  
Regulate Cell Growth ◽  
Epigenetic Processes

Nutrients and metabolic pathways regulate cell growth and cell fate decisions via epigenetic modification of DNA and histones. Another key genetic material, RNA, also contains diverse chemical modifications. Among these, N6-methyladenosine (m6A) is the most prevalent and evolutionarily conserved RNA modification. It functions in various aspects of developmental and disease states, by controlling RNA metabolism, such as stability and translation. Similar to other epigenetic processes, m6A modification is regulated by specific enzymes, including writers (methyltransferases), erasers (demethylases), and readers (m6A-binding proteins). As this is a reversible enzymatic process, metabolites can directly influence the flux of this reaction by serving as substrates and/or allosteric regulators. In this review, we will discuss recent understanding of the regulation of m6A RNA modification by metabolites, nutrients, and cellular metabolic pathways.

scholarly journals The Cellular Senescence Stress Response in Post-Mitotic Brain Cells: Cell Survival at the Expense of Tissue Degeneration

Life ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 229
Author(s):  
Eric Sah ◽  
Sudarshan Krishnamurthy ◽  
Mohamed Y. Ahmidouch ◽  
Gregory J. Gillispie ◽  
Carol Milligan ◽  
...  
Keyword(s):  
Stress Response ◽  
Cell Fate ◽  
Cellular Senescence ◽  
Cell Biology ◽  
Brain Aging ◽  
Complex Stress ◽  
Brain Cell ◽  
Tissue Degeneration ◽  
Cellular Dna

In 1960, Rita Levi-Montalcini and Barbara Booker made an observation that transformed neuroscience: as neurons mature, they become apoptosis resistant. The following year Leonard Hayflick and Paul Moorhead described a stable replicative arrest of cells in vitro, termed “senescence”. For nearly 60 years, the cell biology fields of neuroscience and senescence ran in parallel, each separately defining phenotypes and uncovering molecular mediators to explain the 1960s observations of their founding mothers and fathers, respectively. During this time neuroscientists have consistently observed the remarkable ability of neurons to survive. Despite residing in environments of chronic inflammation and degeneration, as occurs in numerous neurodegenerative diseases, often times the neurons with highest levels of pathology resist death. Similarly, cellular senescence (hereon referred to simply as “senescence”) now is recognized as a complex stress response that culminates with a change in cell fate. Instead of reacting to cellular/DNA damage by proliferation or apoptosis, senescent cells survive in a stable cell cycle arrest. Senescent cells simultaneously contribute to chronic tissue degeneration by secreting deleterious molecules that negatively impact surrounding cells. These fields have finally collided. Neuroscientists have begun applying concepts of senescence to the brain, including post-mitotic cells. This initially presented conceptual challenges to senescence cell biologists. Nonetheless, efforts to understand senescence in the context of brain aging and neurodegenerative disease and injury emerged and are advancing the field. The present review uses pre-defined criteria to evaluate evidence for post-mitotic brain cell senescence. A closer interaction between neuro and senescent cell biologists has potential to advance both disciplines and explain fundamental questions that have plagued their fields for decades.

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