RNA modification enzymes / edited by Jane E. Jackman.

Format:

Book

Contributor:

Jackman, Jane E., editor.

Series:

Methods in enzymology ; Volume 658.

Methods in Enzymology ; Volume 658

Language:

English

Subjects (All):

Catalytic RNA.

RNA Editing.

Nucleotides.

RNA, Transfer.

Medical Subjects:

RNA Editing.

Nucleotides.

RNA, Transfer.

Physical Description:

1 online resource (476 pages)

Edition:

1st ed.

Place of Publication:

Cambridge, MA : Academic Press is an imprint of Elsevier, [2021]

Summary:

RNA Modification Enzymes, Volume 659 in the Methods in Enzymology series, highlights new advances in the field, with this new volume presenting interesting chapters on a variety of related topics, including Locating chemical modifications in RNA sequences through ribonucleases and LC-MS based analysis, Development of RNA modification mapping.

Contents:

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

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

Chapter Three: Facile detection of RNA phospho-methylation in cells and tissues

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

Chapter Four: Quantitative probing of glycosylated queuosine modifications in tRNA

2. Methods

2.1. Total RNA deacylation

2.2. Acid denaturing polyacrylamide gel electrophoresis

2.3. Nonradioactive Northern blot quantification

3. Notes

References.

Chapter Five: CTS tag-based methods for investigating mitochondrial RNA modification factors in Trypanosoma brucei

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

4.2. Reagents and buffers

4.3. Procedure

Chapter Six: Analysis of the epitranscriptome with ion-pairing reagent free oligonucleotide mass spectrometry

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

3.6.1. Agilent´s Qualsoftware

3.6.2. In silico tools and data analysis

4. Notes

Chapter Seven: RNA immunoprecipitation to identify in vivo targets of RNA editing and modifying enzymes

2. Factors to consider when designing a RIP assay

3. Step-by-step method details

3.1. Materials and equipment

4. Troubleshooting

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

Chapter Nine: Analysis of codon-specific translation by ribosome profiling

2. Before you begin

2.1. Key resources table

2.2. Abbreviations

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

8. Optimization and troubleshooting

9. Safety considerations and standards

10. Alternative methods/procedures

Chapter Ten: Partially modified tRNAs for the study of tRNA maturation and function.

Notes:

Description based on print version record.

Description based on publisher supplied metadata and other sources.

ISBN:

9780128235867

0128235861

9780128235850

0128235853

OCLC:

1268546688