Main description:

This detailed volume explores the latest methods that can be used to probe mRNA decay pathways and identify mRNA-binding protein targets as well as miRNA targets. Subjects include metabolic labelling and RNAseq methods for determining RNA decay rates, approaches for discovering RNA-binding protein targets, bioinformatics, miRNA targets and novel components of the miRNA-directed decay pathway, and recently developed approaches for studying nonsense-mediated mRNA decay, among other areas. Written for the highly popular Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, mRNA Decay: Methods and Protocols serves as an ideal guide for molecular biologists, geneticists, and developmental biologists with an interest in understanding how normal development and tissue homeostasis is regulated and how these processes are perturbed in inherited and acquired diseases.

Contents:

1. 5'bromouridine IP Chase (BRIC)-Seq to Determine RNA Half Lives

Toshimichi Yamada, Naoto Imamachi, Rena Mizutani, Katsutoshi Imamura, Yutaka Suzuki, and Nobuyoshi Akimitsu

2. Determining mRNA Decay Rates Using RNA Approach to Equilibrium Sequencing (RATE-Seq)

Farah Abdul-Rahman and David Gresham

3. Metabolic Labeling of Newly Synthesized RNA with 4sU to in Parallel Assess RNA Transcription and Decay

Wei Sun and Wei Chen

4. Measuring mRNA Decay in Budding Yeast Using Single Molecule FISH

Tatjana Trcek, Samir Rahman, and Daniel Zenklusen

5. PAR-CLIP for Discovering Target Sites of RNA-Binding Proteins

Aitor Garzia, Pavel Morozov, Marcin Sajek, Cindy Meyer, and Thomas Tuschl

6. Characterizing mRNA Sequence Motifs in the 3'UTR Using GFP Reporter Constructs

Rene Geissler and Andrew Grimson

7. iCLIP of the PIWI Protein Aubergine in Drosophila Embryos

Bridlin Barckmann, Jeremy Dufourt, and Martine Simonelig

8. Integration of ENCODE RNAseq and eCLIP Data Sets

Jorge Boucas

9. Identifying miRNA Targets Using AgoRIP-Seq

Rebecca Petri and Johan Jakobsson

10. Integrated Analysis of miRNA and mRNA Expression Profiles to Identify miRNA Targets

Zhiming Li and Chi-Meng Tzeng

11. Identifying RISC Components Using Ago2 Immunoprecipitation and Mass Spectrometry

Tingfang Yi

12. Using Tet-Off Cells and RNAi Knockdown to Assay mRNA Decay

Thomas D. Baird and J. Robert Hogg

13. Identifying Cellular Nonsense-Mediated mRNA Decay (NMD) Targets: Immunoprecipitation of Phosphorylated UPF1 Followed by RNA Sequencing (p-UPF1 RIP-Seq)

Tatsuaki Kurosaki, Mainul Hoque, and Lynne E. Maquat

14. Generation of Cell Lines Stably Expressing a Fluorescent Reporter of Nonsense-Mediated mRNA Decay Activity

Nadezhda M. Markina, Anton P. Pereverzev, Dmitry B. Staroverov, Konstantin A. Lukyanov, and Nadya G. Gurskaya

15. Reactivation Assay to Identify Direct Targets of the Nonsense-Mediated mRNA Decay Pathway in Drosophila

Jonathan O. Nelson and Mark M. Metzstein

16. Studying Nonsense-Mediated mRNA Decay in Mammalian Cells Using a Multicolored Bioluminescence-Based Reporter System

Andrew Nickless and Zhongsheng You