Deadenylation, 1st ed. 2024 Methods and Protocols Methods in Molecular Biology Series, Vol. 2723

This volume provides new approaches and technologies into roles of poly(A) metabolism in translation, RNA stability, and quality control of gene expression. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Deadenylation: Methods and Protocols aims to pave the way for future investigations of the complex regulatory networks that control mRNA stability and expression.

1. The reconstitution of human CCR4-NOT from purified proteins and an assay of its deadenylation activity

Yevgen Levdansky and Eugene Valkov

2. In vitro reconstitution of the Drosophila melanogaster CCR4-NOT complex to assay deadenylation

Filip Pekovic and Elmar Wahle

3. Analysis of human mRNA deadenylation complexes via high-resolution gel electrophoresis

Chung-Te Chang

4. Quantitative biochemical analysis of deadenylase enzymes using fluorescence and chemiluminescence-based assays.

Blessing Airhihen, Lorenzo Pavanello, Maryati Maryati, and Gerlof Sebastiaan Winkler

5. A FRET-based assay to quantify enzymatic rates and explore the mechanisms of RNA deadenylases in heterogeneous environments.

Rose Irwin, Robert W. Harkness, and Julie D. Forman-Kay

6. Measuring Poly-Adenosine Tail Length of RNAs by High Resolution Northern Blotting Coupled with RNase H Cleavage

René M. Arvola and Aaron C. Goldstrohm

7. An RNA-ligation-based RACE-PAT assay to monitor poly(A) tail length of mRNAs of interest.

Fabienne Mauxion and Bertrand Séraphin

8. Dissecting the role of the Ccr4-Not deadenylase complex in pluripotency and differentiation

9. Measuring proximity-mediated function of mRNA regulatory proteins by engineered tethering

Breanne M. Hatfield, Chase A. Weidmann, and Kevin M. Weeks

10. Tethered mRNA Amplifier: A Novel Approach to Increase Protein Expression.

Bahareh Torkzaban, Austin Sponaugle, and Jeff Coller

11. RNA binding protein-mediated mRNA deadenylation in mammalian cell extracts.

Wi S. Lai, Stephanie N. Hicks, and Perry J. Blackshear

12. Transcriptome-wide analysis of mRNA adenylation status in yeast using nanopore sequencing.

Pawel S Krawczyk, Agnieszka Tudek, Seweryn Mroczek, and Andrzej Dziembowski

13. Sequencing of transcriptome-wide poly(A) tails by PAIso-seq.

Yusheng Liu, Falong Lu, and Jiaqiang Wang

14. Nano3′RACE: a method to analyze poly(A) tail length and nucleotide additions at the 3′ extremity of selected mRNAs using nanopore sequencing

Pietro Giraudo, Quentin Simonnot, David Pflieger, Jackson Peter, Dominique Gagliardi and Hélène Zuber

15. Quantification of poly(A) tail length and terminal modifications using direct RNA sequencing

Ankit Gupta, Brian N. Papas, Marine Baptissart, and Marcos Morgan

16. Differential poly(A) tail length analysis using nanopore sequencing.

Showkat A. Dar, Sulochan Malla, and Manolis Maragkakis

17. Single-molecule poly(A) tail sequencing (SM-PATseq) using the PacBio platform.

James R. Iben, Tianwei Li, Sandy Mattijssen, and Richard J. Maraia

18. Mathematical models for the poly(A) tail shortening process.

Benedict Fabia, Minju Kim, Jongmin Lim, and Young-suk Lee

Includes cutting-edge methods and protocols

Provides step-by-step detail essential for reproducible results

Contains key notes and implementation advice from the experts