RNA modification enzymes /

Detalles Bibliográficos
Clasificación:Libro Electrónico
Otros Autores: Jackman, Jane E. (Editor )
Formato: Electrónico eBook
Idioma:Inglés
Publicado: Cambridge, MA : Academic Press, 2021.
Colección:Methods in enzymology ; v. 658.
Temas:
Catalytic RNA.
Nucleotides.
Transfer RNA.
RNA Editing
Nucleotides
RNA, Transfer
Ribozymes.
Nucl�eotides.
ARN de transfert.
Catalytic RNA
Internet Resources.
Index not Present.
Acceso en línea:Texto completo
Tabla de Contenidos:
  • Intro
  • RNA Modification Enzymes
  • Copyright
  • Contents
  • Contributors
  • Preface
  • Chapter One: Locating chemical modifications in RNA sequences through ribonucleases and LC-MS based analysis
  • 1. Introduction
  • 2. Characterization of chemical modifications in RNA
  • 2.1. Identity and census of resident modifications
  • 2.2. Locating the chemical modification in the RNA sequence
  • 3. Tools to characterize modified RNA sequence
  • 3.1. Equipment (available through multiple vendors)
  • 3.2. Chemicals
  • 4. Protocols
  • 4.1. RNA hydrolysis to nucleosides
  • 4.2. LC-MS based identification of modifications
  • 4.3. Oligonucleotide generation for location-specific information
  • 4.3.1. Nucleobase-specific ribonucleases
  • 4.3.2. Nucleoside-preferential ribonucleases (alternate approach)
  • 4.3.3. Non-specific ribonucleases
  • 4.4. LC-MS analysis for sequencing the oligonucleotides
  • 4.5. Data analysis
  • 5. Summary
  • Acknowledgment
  • References
  • Chapter Two: Mapping of 7-methylguanosine (mC) RNA modifications by AlkAniline-Seq
  • 1. Introduction
  • 2. Chemical approaches for mapping of mC
  • 2.1. Cleavage of N-glycosidic bond and/or detection of the reverse transcription arrest
  • 2.2. Selective ligation to the 5-phosphate resulting from RNA abasic site
  • 3. Overview of AlkAniline-Seq protocol
  • 3.1. RNA fragmentation step
  • 3.2. De-phosphorylation
  • 3.3. Aniline cleavage
  • 3.4. Adapter ligation and barcoding
  • 3.5. Sequencing
  • 3.6. Data analysis
  • 4. Analysis of tRNA and rRNA in total RNA fraction
  • 4.1. Equipment
  • 4.2. Chemicals
  • 4.3. Consumables and kits for library preparation and sequencing
  • 4.4. Biological material
  • 5. AlkAniline-Seq protocol
  • 5.1. RNA fragmentation in bicarbonate buffer at 95C
  • 5.2. De-phosphorylation by Antarctic phosphatase
  • 5.3. Aniline cleavage.
  • 5.4. Library preparation using NEBNext multiplex small RNA library prep set for Illumina
  • 5.5. Purification of the library using GeneJET PCR purification kit
  • 5.6. Library quantification and quality assessment
  • 5.7. Sequencing of the library
  • 5.8. Bioinformatic analysis
  • 6. Data analysis and interpretation
  • 7. Limitations
  • 8. Summary
  • Acknowledgments
  • References
  • Chapter Three: Facile detection of RNA phospho-methylation in cells and tissues
  • 1. Introduction
  • 1.1. Before you begin
  • 1.1.1. Buffer preparation
  • 1.1.2. Key resources table
  • 1.2. Materials and equipment
  • 1.2.1. Cell culture
  • 1.2.1.1. Equipment
  • 1.2.1.2. Reagents
  • 1.2.2. Protein and RNA extraction
  • 1.2.2.1. Equipment
  • 1.2.2.2. Reagents
  • 1.2.3. Antarctic phosphatase or terminator treatment
  • 1.2.3.1. Equipment
  • 1.2.3.2. Reagents
  • 1.2.4. RNA cleanup
  • 1.2.5. Northern blot
  • 1.2.5.1. Equipment
  • 1.2.5.2. Reagents
  • 1.2.5.3. Alternatives
  • 2. Protocol
  • 2.1. Seeding cells
  • 2.2. Preparing protein and RNA extracts from same cells with the Norgen RNA/Protein Plus kit (Product #48200)
  • 2.3. AP-shift assay for tRNA
  • 2.4. Terminator assay
  • 2.5. RNA clean-up using the Qiagen RNeasy MinElute kit
  • 2.6. Northern blotting
  • 3. Expected outcomes
  • 4. Quantification and statistical analysis
  • 5. Advantages
  • 6. Limitations
  • 7. Optimization and troubleshooting
  • 7.1. Problem
  • 7.2. Potential solution to optimize the procedure
  • 8. Safety considerations and standards
  • 9. Alternative methods/procedures
  • References
  • Chapter Four: Quantitative probing of glycosylated queuosine modifications in tRNA
  • 1. Introduction
  • 2. Methods
  • 2.1. Total RNA deacylation
  • 2.2. Acid denaturing polyacrylamide gel electrophoresis
  • 2.3. Nonradioactive Northern blot quantification
  • 3. Notes
  • Acknowledgments
  • References.
  • Chapter Five: CTS tag-based methods for investigating mitochondrial RNA modification factors in Trypanosoma brucei
  • 1. Introduction
  • 2. Protein affinity purification and in vivo proximity labeling
  • 2.1. In situ CTS-tagging
  • 2.2. Purification of protein and ribonucleoprotein complexes
  • 2.2.1. Equipment
  • 2.2.2. Buffers and reagents
  • 2.2.3. Procedure
  • 2.3. In vivo proximity biotinylation
  • 2.3.1. Equipment
  • 2.3.2. Buffers and reagents
  • 2.3.3. Procedure
  • 3. UV-crosslinking tandem affinity purification sequencing (CTAP-SEQ)
  • 3.1. UV-crosslinking, purification and RNA-Seq library preparation
  • 3.1.1. Equipment
  • 3.1.2. Buffers and reagents
  • 3.1.3. Procedure
  • 3.2. CTAP-SEQ data analysis
  • 3.2.1. Data pre-processing
  • 3.2.1.1. Adaptor trimming
  • 3.2.1.2. Handling unique molecular identifier (UMI)
  • 3.2.1.3. Filtering out nuclear genome-encoded RNAs
  • 3.2.2. Binding sites distribution along RNAs of interest
  • 4. Immunofluorescence imaging of CTS-tagged proteins
  • 4.1. Equipment
  • 4.2. Reagents and buffers
  • 4.3. Procedure
  • 5. Summary
  • Acknowledgments
  • References
  • Chapter Six: Analysis of the epitranscriptome with ion-pairing reagent free oligonucleotide mass spectrometry
  • 1. Introduction
  • 2. Materials
  • 2.1. Cell culture
  • 2.2. RNA isolation
  • 2.3. RNA purification
  • 2.4. PCR and in vitro transcription
  • 2.5. AlkB in vitro assay
  • 2.6. Primers, oligonucleotides and templates
  • 2.7. Digestion
  • 2.8. LC-MS
  • 3. Methods
  • 3.1. General workflow of an oligonucleotide (ON)-MS experiment
  • 3.1.1. Considerations for biological samples (RNA substrates)
  • 3.1.2. Considerations for RNA digestion
  • 3.1.3. Considerations for mass spectrometry
  • 3.2. RNA substrates
  • 3.2.1. Cell culture
  • 3.2.2. T7 in vitro transcribed RNA as substrate or internal standard
  • 3.2.3. Synthetic oligonucleotides.
  • 3.3. Processing of RNA
  • 3.3.1. RNA isolation for native RNA
  • 3.3.2. SEC separation for native tRNA and in vitro transcribed tRNA
  • 3.3.3. RNA in vitro demethylation with AlkB
  • 3.4. RNase T1 digest for ON-MS
  • 3.5. Low- and high-resolution mass spectrometry
  • 3.5.1. Low-resolution QQQ for oligonucleotide mass spectrometry
  • 3.5.2. Comparison of chromatographic set-up for nucleoside-MS and ON-MS
  • 3.5.3. High-resolution orbitrap for ON-MS
  • 3.6. Data analysis
  • 3.6.1. Agilent�s Qualsoftware
  • 3.6.2. In silico tools and data analysis
  • 4. Notes
  • Acknowledgments
  • References
  • Chapter Seven: RNA immunoprecipitation to identify in vivo targets of RNA editing and modifying enzymes
  • 1. Introduction
  • 2. Factors to consider when designing a RIP assay
  • 3. Step-by-step method details
  • 3.1. Materials and equipment
  • 4. Troubleshooting
  • 5. Summary
  • Acknowledgment
  • Reference
  • Chapter Eight: Chemoenzymatic labeling of RNA to enrich, detect and identify methyltransferase-target sites
  • 1. Before you begin
  • 2. Key resources table
  • 3. Materials and equipment
  • 3.1. Equipment
  • 3.2. Materials
  • 3.3. Reagents
  • 3.3.1. Alternatives
  • 4. Step-by-step method details
  • 4.1. Recombinant expression and purification of METTL3-METTL14
  • 4.1.1. Generation of recombinant bacmids in E. coli DH10Bac cells
  • 4.1.2. Cultivation and transfection of Sf21 insect cells
  • 4.1.3. Preparation of V1 baculovirus stock
  • 4.1.4. SDS-PAGE analysis
  • 4.1.5. Small-scale expression test
  • 4.1.6. Large-scale expression
  • 4.1.7. Purification of METTL3-METTL14
  • 4.2. Synthesis of 5-[(R/S)-[(3S)-3-amino-3-carboxy-propyl]prop-2-yn-1-ylselenio]-5-deoxyadenosine (SeAdoYn)
  • 4.2.1. In vitro MTase assay
  • 4.3. Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)
  • 4.3.1. Cleanup via microspin G-25 columns
  • 4.3.2. Enrichment via magnetic beads.
  • 4.3.3. Detection via primer extension assay
  • 4.4. PAGE
  • 5. Expected outcomes
  • 6. Quantification and data analysis
  • 7. Advantages
  • 8. Limitations
  • 9. Optimization and troubleshooting
  • 9.1. Problematic step: Generation of recombinant bacmids in E. coli DH10Bac cells
  • 9.2. Problematic step: Synthesis of SeAdoYn
  • 9.3. Problematic step: CuAAC
  • 10. Safety considerations and standards
  • 11. Alternative methods/procedures
  • 11.1. Metabolic labeling
  • 11.2. Extraction of total RNA from HeLa cells
  • 11.3. Detection via fluorescence
  • 11.4. End-point kinetics measurements
  • Acknowledgments
  • References
  • Chapter Nine: Analysis of codon-specific translation by ribosome profiling
  • 1. Introduction
  • 2. Before you begin
  • 2.1. Key resources table
  • 2.2. Abbreviations
  • 3. Step-by-step method details
  • 3.1. Sample harvesting and RNase I digestion
  • 3.1.1. HEK293T cells: Cell harvesting and extract preparation (Video 1)
  • 3.1.2. HEK293T cells: RNA digestion
  • 3.1.3. Yeast: Cell harvesting (Video 2)
  • 3.1.4. Yeast: cell lysis (Video 3)
  • 3.1.5. Yeast: Cell extract preparation and RNA digestion (Video 4)
  • 3.2. Gradient ultracentrifugation and fractionation (Video 5)
  • 3.3. RNA isolation from monosome fraction (Video 6)
  • 3.4. Size selection of ribosome-protected fragments (RPF) by gel electrophoresis (Video 7)
  • 3.5. Dephosphorylation (Video 8)
  • 3.6. Adapter ligation
  • 3.7. Reverse transcription (Video 9)
  • 3.8. cDNA circularization (Video 10)
  • 3.9. Test PCR (Video 11)
  • 3.10. Library PCR
  • 4. Expected outcomes
  • 5. Quantification and statistical analysis
  • 5.1. Data processing and quality control
  • 5.2. Differential translation analysis
  • 6. Advantages
  • 7. Limitations
  • 8. Optimization and troubleshooting
  • 9. Safety considerations and standards
  • 10. Alternative methods/procedures
  • References.

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