Main description:

This Open Access volume provides comprehensive reviews and describes the latest techniques to study eukaryotic ribosome biogenesis. For more than 50 years ribosomes are a major research topic. Our knowledge about ribosome biogenesis and function such as transcription, mRNA modification, and translation was the sine qua non for developing the powerful RNA-based vaccines against RNA-viruses causing the world-threatening Covid-19 pandemia. The chapters in this book are organized into six parts. Part One discusses a comparative survey about the unity and diversity of ribosome biogenesis in pro- and eukaryotic cells. Part Two deals with the genomic organization of eukaryotic rDNA and the role of RNA polymerase I in ribosomal RNA transcription. Part Three explores in vitro methods to study RNA polymerase I structure and its function, and Part Four analyzes the nucleo-cytoplasmic transport of assembled ribosomes and RNP complexes. Part Five covers modifications that increase the complexity of rRNAs, and Part Six provides readers with a review of eukaryotic translation and - for the first time - describes a new method to analyze translation in vitro. 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 tips on troubleshooting and avoiding known pitfalls.
Cutting-edge and comprehensive, Ribosome Biogenesis: Methods and Protocols is a valuable resource for scientists and researchers interested in learning more about the increasing importance of in vitro RNA-technologies.

Contents:

Dedication......
Acknowledgments...
Preface...
Table of Contents...
Contributing Authors...

Part I Ribosome Biogenesis

1. A Comparative Perspective on Ribosome Biogenesis: Unity and Diversity across the Tree of Life
Michael Juttner and Sebastien Ferreira-Cerca

Part II Genomic Organization

2. Establishment and Maintenance of Open Ribosomal RNA Gene Chromatin States in Eukaryotes
Christopher Schachner, Philipp E. Merkl, Michael Pilsl, Katrin Schwank, Kristin Hergert, Sebastian Kruse, Philipp Milkereit, Herbert Tschochner, and Joachim Griesenbeck

3. Analysis of Yeast RNAP I Transcription of Nucleosomal Templates In Vitro
Philipp E. Merkl, Christopher Schachner, Michael Pilsl, Katrin Schwank, Kristin Hergert, Gernot Langst, Philipp Milkereit, Joachim Griesenbeck, and Herbert Tschochner

Part III RNA Polymerases

4. Specialization of RNA Polymerase I in Comparison to Other Nuclear RNA Polymerases of Saccharomyces cerevisiae
Philipp E. Merkl, Christopher Schachner, Michael Pilsl, Katrin Schwank, Catharina Schmid, Gernot Langst, Philipp Milkereit, Joachim Griesenbeck, and Herbert Tschochner

5. Structural Studies of Eukaryotic RNA Polymerase I using Cryo-Electron Microscopy
Michael Pilsl and Christoph Engel

6. Preparation of RNA Polymerase Complexes for their Analysis by Single Particle Cryo-Electron Microscopy
Michael Pilsl, Florian B. Heiss, Gisela Poell, Mona Hoecherl, Philipp Milkereit, and Christoph Engel

Part IV Ribosome Assembly, Transport and RNP Complexes

7. Eukaryotic Ribosome Assembly and Nucleo-Cytoplasmic Transport
Michaela Oborska-Oplova, Ute Fischer, Martin Altvater, and Vikram Govind Panse

8. Tethered MNase Structure Probing as Versatile Technique for Analyzing RNPs using Tagging Cassettes for Homologous Recombination in Saccharomyces cerevisiae
Fabian Teubl, Katrin Schwank, Uli Ohmayer, Joachim Griesenbeck, Herbert Tschochner, and Philipp Milkereit

Part V RNA Modification

9. Chemical Modifications of Ribosomal RNA
Sunny Sharma and Karl-Dieter Entian

10. In Vitro Selection of Deoxyribozymes for the Detection of RNA Modifications
Anam Liaqat, Maksim V. Sednev, and Claudia Hoebartner

11. Mapping of the Chemical Modifications of rRNAs
Jun Yang, Peter Watzinger, and Sunny Sharma

12. Non-Radioactive In Vivo Labelling of RNA with 4-thio-uracil
Christina Braun, Robert Knuppel, Jorge Perez-Fernandez, and Sebastien Ferreira-Cerca

Part VI Translation

13. Translation Phases in Eukaryotes
Sandra Blanchet and Namit Ranjan

14. Differential Translation Activity using Bio-Orthogonal Non-Canonical Amino Acids Tagging (BONCAT) in Archaea
Michael Kern and Sebastien Ferreira-Cerca

15. Thermofluor-Based Analysis of Protein Integrity and Ligand Interactions
Sophia Pinz, Eva Doskocil, and Wolfgang Seufert

16. In Vitro Assembly of a Fully Reconstituted Yeast Translation System for Studies of Initiation and Elongation Phases of Protein Synthesis
Sandra Blancher and Namit Ranjan