Polyadenylation: Methods and Protocols (eng)

PART I: SECTION I: 3'-END PROCESSING IN EUKARYOTES

1. 3' end Formation and Regulation of Eukaryotic mRNAs

Thomas J. Sweet and Donny D. Licatalosi

PART II: I-A General Methods

2. Poly(A) Polymerase-based Poly(A) Length Assay

Deepak P. Patil, Baskar Bakthavachalu, and Daniel R. Schoenberg

3. Using Klenow Mediated Extension to Measure Poly(A)-tail Length and Position in the Transcriptome

Man C. Lee, Amrei Jänicke, and Traude H. Beilharz

4. Detection of Uridylated mRNAs

François M. Sement, Dominique Gagliardi

5. Cytoplasmic Polyadenylation Assays

Olga Coll, Ana Villalba, Fátima Gebauer

6. In vitro Analysis of Transcriptional Activators and Polyadenylation

Takashi Nagaike and James L. Manley

7. Fluorescence Detection of Polyadenylation Reaction through the Coordination of Adensoine₂-coralyne-adensoine₂

Jia-Hui Lin and Wei-Lung Tseng

8. Small Molecule Recognition of Poly(A)

Özgül Persil Çetinkol

9. The Stem-loop Luciferase Assay for Polyadenylation (SLAP) Method for Determining CstF-64-dependent Polyadenylation Activity

J. Andrew Hockert and Clinton C. MacDonald

PART III: I-B Alternative Polyadenylation

I-B-1 Prediction of Polyadenylation Sites

10. Accurate Mapping of Cleavage and Polyadenylation Sites by 3' Region Extraction and Deep Sequencing

Mainul Hoque, Wencheng Li, and Bin Tian

11. Polyadenylation Site Prediction using PolyA-iEP Method

Ioannis Kavakiotis, George Tzanis, and Ioannis Vlahavas

12. Detecting Alternative Polyadenylation from Microarray Data

Antonio Lembo and Paolo Provero

Part IV: I-B-2 Large Scale/Genome Wide Analysis of mRNA Polyadenylation and Application of the Next Generation DNA Sequencing in Polyadenylation Study

13. Quantitative Polyadenylation Site Mapping with Single-molecule Direct RNA Sequencing

Fatih Ozsolak

14. RHAPA: A New Method to Quantify Alternative Polyadenylation

Ashley L. Cornett and Carol S. Lutz

15. Multiplex Analysis of PolyA-linked Sequences (MAPS): An RNA-seq Strategy to Profile poly(A+) RNA

Yu Zhou, Hai-Ri Li, Jie Huang, Ge Jin, and Xiang-Dong Fu

16. Global and Quantitative Profiling of Polyadenylated RNAs using PAS-seq

Chengguo Yao and Yongsheng Shi

17. High-throughput Sequencing of RNA Isolated by Crosslinking and Immunoprecipitation (HITS-CLIP) to Determine Sites of Binding of CstF-64 on Nascent RNAs

Petar N. Grozdanov and Clinton C. MacDonald

Part V: BACTERIA AND ORGANELLES

18. Polyadenylation in Bacteria and Organelles

Joanna Rorbach, Agnieszka J. Bobrowicz, Sarah Pearce, and Michal Minczuk.

19. In vivo Analysis of Polyadenylation in Prokaryotes

Bijoy K. Mohanty and Sidney R. Kushner

20. Polyadenylation of RNA in E. coli: RNA Polymerase-associated (rA)n-synthetic Activities

Grant A. Encalade and Maxim V. Sukhodolets

21. Analysis of the Human Mitochondrial Transcriptome using Directional Deep sequencing and Parallel Analysis of RNA Ends

Oliver Rackham and Aleksandra Filipovska

22. Measurement of Mitochondrial RNA Stability by Metabolic Labeling of Transcripts with 4-Thiouridine

Lukasz S. Borowski and Roman J. Szczesny

Part VI: DEADENYLATION AND DECAY

23. Deadenylation and its Regulation in Eukaryotic Cells

Xiaokan Zhang, Frida E. Kleiman, and Emral Devany

24. Assaying mRNA Deadenylation in vitro

Mandy Jeske, Claudia Temme, and Elmar Wahle

25. Assaying mRNA Deadenylation in vivo

Claudia Temme and Elmar Wahle

26. In vitro Analysis

In Polyadenylation: Methods and Protocols, expert researchers in the field detail many of the protocols which are now commonly used to study polyadenylation. Focusing on recent advances in the fast-moving polyadenylation filed, that has recently been recognized as a key contributor to the complexity of mammalian 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.