RNA Enzymology and Technology.

Format:

Book

Author/Creator:

Liu, Kathy.

Contributor:

Kathy Fange Liu

Series:

Methods in Enzymology Series

Methods in Enzymology Series ; v.Volume 726

Language:

English

Subjects (All):

Catalytic RNA.

RNA editing.

Physical Description:

1 online resource (0 pages)

Edition:

1st ed.

Place of Publication:

Chantilly : Elsevier Science & Technology, 2026.

Summary:

This volume of Methods in Enzymology brings together a comprehensive collection of experimental approaches and mechanistic insights into enzymes work on RNAs--molecular catalysts at the heart of RNA metabolism, regulation, and therapeutics.

Contents:

Front Cover

Series Page

Methods in Enzymology

Copyright

Contents

Contributors

Section 1: RNA enzymes in transcription, RNA processing, and translation regulation

Chapter One: Unmasking a hidden code during transcription: A practical strategy to reveal Thr4 phosphorylation in RNA polymerase II

1 Introduction

2 Approach

2.1 Ssu72 can dephosphorylate Ser5 CTD but not at Thr4

2.2 Before you begin

2.3 Key resources table

2.4 Materials and equipment

2.5 Specificity of Ssu72 with MALDI-TOF

2.6 Safety considerations and standards

2.7 Troubleshooting and experimental optimization

2.8 Experimental results and expected outcomes

3 Activity of Ssu72 from various organisms

3.1 Before you begin

3.2 Key resources table

3.3 Materials and equipment

3.4 Generate absorbance standard curve

3.5 Set up pNPP assay for Ssu72 phosphatase

3.6 Advantages and limitations

3.7 Troubleshooting and optimization

3.8 Experimental results and expected outcomes

4 Purification of Ssu72/Symplekin

4.1 Before you begin

4.2 Key resources table

4.3 Materials and equipment

4.4 Phosphatase Ssu72 purification

5 Ssu72 optimization in cell lysate for unmasking pThr4

6 ChIP-seq analysis with Ssu72/Symplekin phosphatase treatment

6.1 Before you begin

6.2 Key resources table

6.3 Materials and equipment

6.4 Cross-link cells, sonication and IP

7 CTD Thr4 phosphorylation profile in human cells

8 Limitation of phosphatase treatment method to unmask the flanking phosphorylation

9 Conclusion

Funding

References

Chapter Two: In Vitro transcriptase: Methods for studying XNA synthesis

2 Key resources table

3 Buffers and solutions

4 SFM 5-7 polymerase expression and purification

4.1 Transformation of plasmid into BL21 (DE3) competent E. coli.

4.1.1 Timing: 2 hr

4.2 Start pre-culture

4.2.1 Timing: 30 min

4.3 Scale-up culture and express SFM 5-7 polymerase

4.3.1 Timing: 4-6 hr

4.4 Harvest cells

4.4.1 Timing: 1 hr

4.5 SFM 5-7 polymerase purification

4.5.1 Timing: 8-10 hr

4.6 Quality control by SDS-PAGE analysis

4.6.1 Timing: 4 hr

5 In Vitro transcription using SFM 5-7 polymerase

5.1 Primer design and preparation

5.1.1 Timing: 5-10 min

5.2 In vitro transcription reaction

5.2.1 Timing: 3 hr

5.3 Monitoring in vitro transcription reaction by urea-PAGE

Chapter Three: Enzymes in high-throughput RNA sequencing: Applications and challenges

2 Comparison of recent RT enzymes for read-through

3 Induro-tRNAseq - a methodology to monitor genome-wide tRNA profiles

4 Materials, general method, and statistical analysis

4.1 Deacylation of tRNA, 3′-end repair, oxidation, and β-elimination

4.2 Ligation of 3′-RNA/DNA adaptor

4.3 Reverse transcription

4.4 Polyacrylamide gel electrophoresis

4.5 CircLigase reaction

4.6 PCR

4.7 Analysis of PCR products

5 Methods

5.1 RNA input

5.2 Deacylation and 3′-end repair of tRNA

5.3 Oxidation and β-elimination before deacylation and 3′-end repair

5.4 Ligation of 3′-RNA/DNA adaptor

5.5 Reverse transcription

5.6 Gel purification of cDNA

5.7 CircLigase reaction

5.8 PCR amplification of cDNA

5.9 Gel electrophoresis of amplified cDNA

5.10 Analysis of PCR products

6 Notes

Appendix A. Supporting information

Chapter Four: An smFRET assay to probe the impact of antibiotics on intersubunit rotation in eukaryotic ribosomes

2 Methods

2.1 General considerations

2.1.1 Notes/additional considerations

2.2 Bacterial and yeast strains

2.3 Buffers

3 Yeast ribosome preparation.

3.1 Purification of 40S and 60S ribosomal subunits

3.1.1 Procedure

3.1.2 Notes/considerations

3.2 Ribosome labelling

3.2.1 Procedure

3.2.2 Notes/considerations

3.3 Subunit association

4 eEF1A and eEF1Bα purification

4.1 eEF1A purification

4.2 eEF1Bα expression and purification

5 mRNA and tRNA preparations

5.1 MFY mRNA transcription and purification

5.2 Aminoacylation and N-acetylation of E.coli tRNAMet

5.3 Aminoacylation of yeast tRNAPhe

6 Assembly of pre-translocation 80S ribosomes

7 Slide preparation and assembly

7.1 Slide preparation

7.1.1 Procedure

7.1.2 Notes/considerations

7.2 Sample chamber assembly

8 smFRET measurements

8.1 smFRET data collection

8.1.1 Procedure

8.1.2 Notes/considerations

9 smFRET data analysis

10 Results

11 Conclusions

Acknowledgements

Author contributions

Chapter Five: A non-radioactive method to detect and measure 48S initiation complex and 80S ribosome formation in vitro

2 Before you begin

3 Key resources table

4 Materials and equipment

5 Step-by-step method details

5.1 Day 1 - In vitro transcription of capped and polyadenylated reporter mRNAs

5.2 Day 2, Part 1 - In vitro translation trapping 48S initiation complexes or 80S ribosomes

5.3 Day 2, Part 2 - Sucrose gradient ultracentrifugation of in vitro translation reactions

5.4 Day 3 - RNA extraction of fractions and RT-qPCR

6 Expected outcomes

7 Quantification and statistical analysis

8 Advantages

9 Limitations

10 Optimization and troubleshooting

10.1 Low yield from in vitro transcription

10.2 Low levels of 48S initiation complex formation

10.3 Low levels of 80S ribosome formation

10.4 Smaller change in 48S and 80S complex levels between controls and experimental conditions than expected.

11 Safety considerations and standards

Section 2: Enzymes in RNA Silencing and Regulation

Chapter Six: Bioinformatic identification of regulatory feedback motifs within RNAi pathways using multi-omics datasets

2 Materials

2.1 General equipment

2.2 General materials

3 Methods

3.1 Overview of paired small RNA-seq and mRNA-seq for regulatory feedback detection

3.2 Preparation of reagents

3.3 Purification of size-selected small RNAs

3.4 Optional 5′ RNA polyphosphatase treatment

3.5 Small RNA-seq library preparation

3.6 Purification of poly adenylated RNAs and mRNA library preparation

3.7 Paired small RNA-seq and mRNA-seq analysis for regulatory feedback detection

4 Notes

Chapter Seven: Induro-seq to analyze subcellular enrichment of small RNAs

2.1 Timing: 1-7 days

4.1 Materials

4.2 Equipment

5.1 Overview

5.2 Subcellular fractionation of mammalian cells and RNA extraction

5.3 (Optional steps) validate subcellular fractionation by RT-qPCR

5.4 Small RNA library preparation with Induro RT and spike-ins

6 Data analysis and expected outcomes

7 Advantages

8 Limitations

Section 3: Enzymes in RNA-Based Technologies

Chapter Eight: Preparation of enzymes and libraries for MapID-tRNA-seq to identify chemical modifications in human tRNAs

1.1 tRNA sequences in humans

1.2 Chemical modifications on tRNAs

1.3 Challenges in mapping modifications in human tRNAs

2 Expression and purification of RT-1306 and AlkB for MapID-tRNA-seq

2.1 Materials and equipment

2.2 Stock solutions.

2.3 Buffers for protein purification and characterization

2.4 Expression, purification, and characterization of RT-1306

2.5 Expression, purification, and characterization of AlkB

3 tRNA purification from mammalian cells

3.1 Reagents and chemicals

3.2 Equipment and supplies

3.3 Total RNA extraction via TRIzol

3.4 tRNA purification by denaturing PAGE gel

4 MapID-tRNA-seq library preparation

4.1 Reagents and chemicals

4.3 Supplies

4.4 Treatment of tRNAs and library preparation

4.5 Example of trouble shooting during library preparation

5 Identification of m1A and m3C assisted by tRNA MapID

5.1 Equipment and software

5.2 Processing and alignment of tRNA-seq raw data

5.3 Initial identification of m1A and m3C modifications by mutation signature

5.4 MapID-assisted mutation pattern analysis

Section 4: RNA Modification and Structural Analysis

Chapter Nine: m6A Immunoprecipitation from Ribosome-Bound mRNA

1 Theory

2.1 Buffers required

3 Materials and equipment

3.1 Materials/reagents

3.2 Equipment

4 Step-by-step method details

4.1 Sample collection and lysis

4.2 Polysome profile sample loading and ultracentrifugation

4.3 Acquisition and pooling of polysome fractions

4.4 RNA extraction from lysates

4.5 RNA extraction from polysome fractions

4.6 DNase digestion

4.7 mRNA pulldown

4.8 Luciferase RNA spike-ins

4.9 mRNA fragmentation (skip for qPCR)

4.10 Immunoprecipitation of m6A-modified transcripts

5 Expected outcomes &amp

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&amp

quantification

6 Advantages

7 Limitations

8 Optimization and troubleshooting

9 Alternative methods/procedures

Funding sources

Author Contributions

References.

Chapter Ten: Synthesis and biochemical studies of N3-methylcytidine(m3C), N4-methylcytidine (m4C) and N4, N4-dimethylcytidine (m42C) modified RNAs.

Notes:

Description based on publisher supplied metadata and other sources.

Part of the metadata in this record was created by AI, based on the text of the resource.

ISBN:

0-443-43133-7

0-443-43132-9

9780443431333

OCLC:

1559218934