Ribosome Biogenesis 1st Edition by Karl Dieter Entian ISBN B0B62XSPSW 9781071625019

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ISBN 10: B0B62XSPSW
ISBN 13: 9781071625019
Author: Karl Dieter Entian

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 increasethe 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.

Ribosome Biogenesis 1st Edition Table of contents:

Part I: Ribosome Biogenesis
Chapter 1: A Comparative Perspective on Ribosome Biogenesis: Unity and Diversity Across the Tree of Life
1 In Search of Unity?
2 Ribosome Biogenesis
2.1 Once Upon a Time Ribosome Basic Facts
3 The Ribosome Assembly Process
4 Facilitating Ribosome Assembly
5 Processing and Modifications of Ribosomal RNA
6 Learning from Organelle Ribosome Biogenesis?
7 Concluding Remarks
References
Part II: Genomic Organization
Chapter 2: Establishment and Maintenance of Open Ribosomal RNA Gene Chromatin States in Eukaryotes
1 Introduction
2 Visualization of rRNA Transcription
2.1 Actively Transcribed rRNA Genes Are Prominent Structures in Nuclear Chromatin Spreads
2.2 rRNA Genes Transcribed by RNAP I Are Nucleosome Depleted
2.3 rRNA Genes Coexist in At Least Two Different Chromatin States
2.4 HMG-Box Proteins Are Architectural Components of Open rRNA Gene Chromatin States
2.5 Molecular Requirements to Establish the Open rRNA Gene Chromatin State
3 Conclusions
References
Chapter 3: Analysis of Yeast RNAP I Transcription of Nucleosomal Templates In Vitro
1 Introduction
2 Materials
2.1 Preparation of Tailed Templates
2.2 In Vitro Nucleosome Assembly
2.3 Purification and Restriction Enzyme Digest of Native Yeast Chromatin
2.3.1 Preparation of Cellular Lysates
2.3.2 Affinity Chromatography of Native Chromatin Domains
2.3.3 Restriction Enzyme Digest of Purified Native Chromatin Domains
2.3.4 DNA Analysis
2.4 Purification of RNAPs I, II, and III from S. cerevisiae
2.5 In Vitro Transcription of Chromatin Templates
3 Methods
3.1 Preparation of Tailed Templates
3.2 In Vitro Nucleosome Assembly
3.3 Purification and Restriction Enzyme Digest of Native Yeast Chromatin
3.3.1 Preparation of Cellular Lysates
3.3.2 Affinity Chromatography
3.3.3 Restriction Enzyme Digest of Purified Native Chromatin Domains
3.3.4 DNA Analysis
3.4 Purification of RNAPs I, II, and III from S. cerevisiae
3.5 In Vitro Transcription of Chromatin Templates
3.5.1 Reaction Setup and RNA Extraction
3.5.2 Denaturing Gel Electrophoresis and Visualization of Radiolabeled Transcripts
3.5.3 Transcript Quantification
4 Notes
References
Part III: RNA Polymerases
Chapter 4: Specialization of RNA Polymerase I in Comparison to Other Nuclear RNA Polymerases of Saccharomyces cerevisiae
1 RNA Polymerase I Has Only One Essential Genomic Target
2 RNA Polymerase I Contains Additional Subunits Resembling Transcription Factors of RNAP II
3 Nuclear RNAPs Transcribe Chromatin Templates
References
Chapter 5: Structural Studies of Eukaryotic RNA Polymerase I Using Cryo-Electron Microscopy
1 Cryo-Electron Microscopy: The New Standard in Transcription Research
2 Pol I Specific Subunits Resemble Built-in Transcription Factors
3 The Pol I Transcription Cycle Visualized In Vitro
4 Outlook
References
Chapter 6: Preparation of RNA Polymerase Complexes for Their Analysis by Single-Particle Cryo-Electron Microscopy
1 Introduction
2 Materials
2.1 Assembly of Pol I Complexes
2.1.1 Materials
2.1.2 Buffers
2.2 Negative Staining
2.2.1 Materials
2.3 Preparation of Ultrathin Carbon Film Coated Grids
2.4 Cryo-Grid Preparation
2.5 Cryo-Electron Microscopy
3 Methods
3.1 Assembly of Pol I Complexes
3.2 Negative Staining
3.3 Preparation of Ultrathin Carbon Film
3.4 Carbon Film Transfer onto EM Grids
3.5 Cryo-Grid Preparation
3.6 Cryo-Electron Microscopy
4 Notes
References
Part IV: Ribosome Assembly, Transport and RNP Complexes
Chapter 7: Eukaryotic Ribosome assembly and Nucleocytoplasmic Transport
1 Introduction
2 Materials, Reagents, and Yeast Media
2.1 Material (Listed in Alphabetical Order)
2.2 Reagents (Listed in Alphabetical Order)
2.3 Yeast Media
3 Pre-ribosome Isolation for Single Particle Cryo-Electron Microscopy
3.1 Preparing Yeast Cells for Cryogenic Lysis (Modified from Rout Lab Protocol: Harvesting Cells and Making Yeast Noodles)
3.1.1 Buffers and Solutions
3.1.2 Yeast Cell Preparation
3.1.3 Cryogenic Lysis of Yeast Cells (See Also Note 2) (Modified from Rout Lab Protocol: Cryogenic Lyses of yeast Cells)
3.1.4 Isolating Pre-ribosomes Using Magnetic Beads (Modified from Oeffinger et al. 2007)
Buffers and Solutions
Method (See Also Notes 5-8)
3.2 Nuclear Import Assays for Ribosomal Proteins
3.2.1 A. RanGTP Mediated Disassembly of a Pse1-Slx9 Complex
Buffers and Solutions
Method
3.2.2 Tsr2 Mediated Disassembly of a Kap123-eS26FLAG Complex (See Also Notes 9-14)
3.2.3 Cell Biological Assays for r-protein Nuclear Import
3.3 Nuclear Export Assays for Pre-ribosomes
3.3.1 Monitoring Nuclear Export of Pre-ribosomes (See Also Notes 15 and 16)
Method
3.3.2 Reconstitution of Crm1-Nuclear Export Complexes In Vitro
Buffers and Solutions
Method (See Also Notes 17-21)
3.3.3 FG-Repeat Binding Assay
Buffers and Solutions
Method (See Also Notes 22-28)
3.4 Genetic Analyses of Nuclear Export
4 Conclusions
5 Notes
References
Chapter 8: Tethered MNase Structure Probing as Versatile Technique for Analyzing RNPs Using Tagging Cassettes for Homologous R…
1 Introduction
2 Materials
2.1 Template Plasmids for PCR Based Amplification of Tagging Cassettes
2.2 Generation by PCR of Tagging Cassettes for Homologous Recombination
2.3 Transformation of Competent Yeast Cells and Screening for Positive Clones
2.4 Yeast Culture and Preparation of Cellular Extracts
2.5 Purification of Preribosomal Particles from Cellular Extracts and Induction of MNase Cleavage
3 Methods
3.1 Primer Design and Choice of the Template Plasmid for PCR Reactions
3.2 Generation of PCR-Based Tagging Cassettes for Homologous Recombination
3.3 Transformation of Competent Yeast Cells and Screening for Positive Clones
3.4 Yeast Cell Culture and Preparation of Cellular Extracts
3.5 Purification of RNPs from Cellular Extracts and Activation of MNase
4 Notes
References
Part V: RNA Modification
Chapter 9: Chemical Modifications of Ribosomal RNA
1 Introduction
2 Ribose Methylation
2.1 C/D Box snoRNPs
3 Pseudouridylation
3.1 H/ACA snoRNPs
4 Base Modifications
4.1 Base Methylation
4.2 Methyl transferases
4.3 N4- Acetylation of Cytidine (ac4C)
4.4 3-Amino carboxy propylation acp
5 Base Modifications of 18S rRNA
5.1 Dim1 Catalyzes m62A1781 and m62A1782 dimethylation
5.2 Bud23 Catalyzes m7G1575 Methylation
5.3 Nep1 (Emg1) and Tsr3 Catalyze Ψ1191 Modification (m1 acp3Ψ1191)
5.4 Kre33/NAT10 Catalyzes ac4C1280 and ac4C1773 Acetylation of the 18S rRNA in a snoRNA Dependent Manner
6 Base Modifications of 25S rRNA
6.1 Rrp8 (Bmt1) Catalyzes m1 A 645 Methylation
6.2 Bmt2 Catalyzes A2142 Methylation (m1A2142)
6.3 Rcm1 (Bmt3) and Nop2 (Bmt4) Catalyze m5C2278 and m5C2870 Methylation
6.4 Bmt5 and Bmt6 Catalyze m3U2634 and m3U2843 Methylation
6.5 25S rRNA of Yeast Does Not Contain any m5U Residues
References
Chapter 10: In Vitro Selection of Deoxyribozymes for the Detection of RNA Modifications
1 Introduction
2 Materials
2.1 Oligonucleotides
2.2 Denaturing Polyacrylamide Gel Electrophoresis
2.3 In Vitro Selection
2.3.1 Phosphorylation of RNA Selection Substrates
2.3.2 Splint Ligation
2.3.3 Selection Step
2.3.4 PCR Amplification
2.3.5 Kinetic Characterization of Deoxyribozymes
3 Methods
3.1 DNA Library Design
3.2 Denaturing Polyacrylamide Gel Electrophoresis
3.3 In Vitro Selection
3.3.1 Phosphorylation of RNA Selection Substrates
3.3.2 Splint Ligation of RNA Substrate to Deoxyribozyme Selection Pool
3.3.3 DNA-Catalyzed Cleavage of DNA-RNA Hybrids (Key Selection Step)
3.3.4 PCR Amplification of Active DNA Enzymes
PCR-I
PCR-II
3.3.5 Continuation of Selection and Identification of Deoxyribozyme Sequences
3.4 Kinetic Characterization of Deoxyribozymes
4 Notes
References
Chapter 11: Mapping of the Chemical Modifications of rRNAs
1 Introduction
2 Materials
2.1 Isolation of Intact 18S and 25S rRNA
2.2 Mung Bean Nuclease Protection Assay
2.3 Quantitative Reversed Phase High-Performance Liquid Chromatography (qRP-HPLC)
3 Methods
3.1 Isolation of Intact 18S and 25S rRNA
3.2 Mung Bean Nuclease Protection assay (MBN Assay)
3.2.1 Hybridization
3.2.2 Mung Bean Nuclease Digestion
3.2.3 Purification of the rRNA Fragments
3.2.4 Mapping of Chemical Modifications to a Single Nucleotide Resolution
3.3 Quantitative Reversed Phase High-Performance Liquid Chromatography (qRP-HPLC)
3.3.1 Preparation of Nucleosides
3.3.2 RP-HPLC
3.3.3 Quantification of the Modified Nucleosides
4 Notes
References
Chapter 12: Non-radioactive In Vivo Labeling of RNA with 4-Thiouracil
1 Introduction
2 Materials
2.1 Microbiological Cultures
2.1.1 Strains
2.1.2 Haloferax Volcanii Enhanced Casamino Acids (Hv-Ca+)
2.1.3 Saccharomyces cerevisiae Minimal Medium (See Note 1)
2.2 Chemical Reagents and Materials for RNA Extraction
2.3 Chemical Reagents for Biotinylation of 4TU-Labeled RNA
2.4 Separation and Immobilization of RNA
2.4.1 Agarose Gel Electrophoresis and Capillary Transfer
2.4.2 Polyacrylamide Gel Electrophoresis and Electro Transfer
2.5 Detection of 4-TU Labeled RNA
3 Methods (See Notes 4 and 5)
3.1 Microbiological Methods (See Note 6)
3.1.1 Haloferax volcanii Medium and Cultivation
3.1.2 Saccharomyces cerevisiae Medium and Cultivation
3.2 In Vivo Pulse Labeling with 4-TU (See Note 9)
3.2.1 Uracil and 4-Thiouracil Stocks
3.2.2 Haloferax volcanii Labeling
3.2.3 Saccharomyces cerevisiae Labeling
3.3 RNA Extraction
3.4 4-TU-Labeled RNA Biotinylation
3.5 Separation and Immobilization of RNA (See Note 11)
3.5.1 Preparation of Denaturing Agarose Gel
3.5.2 Preparation of Denaturing Polyacrylamide Gel
3.5.3 Northern Blotting (Agarose Gel)
3.5.4 Northern Blotting (Polyacrylamide Gel)
3.5.5 Detection of Biotinylated RNA
4 Notes
References
Part VI: Translation
Chapter 13: Translation Phases in Eukaryotes
1 Introduction
2 Initiation
3 Elongation
4 Termination
5 Ribosome Recycling
References
Chapter 14: Differential Translation Activity Analysis Using Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) in Archaea
1 Introduction
2 Materials
2.1 Microbiological Cultures
2.1.1 Strains
2.2 Haloferax volcanii Minimal Medium (Hv-min)
2.3 Noncanonical Amino Acid Pulse Reagents
2.4 Cell Lysis
2.5 Protein Reduction
2.6 Acetone Precipitation of Total Proteins
2.7 Protein Alkylation
2.8 Click-Chemistry Reagents-Strain-Promoted Alkyne-Azide Cycloaddition (SPAAC)
2.9 Methanol-Chloroform Extraction of Proteins
2.10 Affinity Purification
2.11 1D/2D Gel Electrophoresis and Detection
3 Methods (Workflow Is Summarized in Fig. 1c)
3.1 Microbiological Methods
3.1.1 Haloferax volcanii Media and Cultivation
3.2 In Vivo Pulse Labeling with L-AHA
3.3 Protein Extraction
3.4 Click-Chemistry
3.4.1 Reduction
3.4.2 Acetone Precipitation
3.4.3 Alkylation
3.4.4 Click-Chemistry: Strain Promoted Alkyne-Azide Cycloaddition (SPAAC)
3.5 Methanol-Chloroform Extraction
3.6 Affinity Purification of Biotinylated Protein
3.6.1 Sample Preparation
3.6.2 Streptavidin Beads Preparation
3.6.3 Affinity Purification
3.6.4 Elution (See Note 9) (an Exemplary Result Is Provided in Fig. 3)
Fast-Elution for Gel Electrophoresis
Competitive Elution for Downstream Processing
3.7 Gel Electrophoresis and Detection
3.7.1 SDS-PAGE
3.7.2 2D Gel Electrophoresis (an Exemplary Result Is Provided in Fig. 4)
Isoelectric Focusing (See Note 2)
Second Dimension SDS-PAGE
3.8 In-Gel Detection (Exemplary Results Are Provided in Figs. 2 and 4)
3.9 Detection of Affinity Purified L-AHA-Labeled Proteins
4 Notes
References
Chapter 15: Thermofluor-Based Analysis of Protein Integrity and Ligand Interactions
1 Introduction
2 Materials
3 Methods
3.1 Optimization of Protein Amount and SYPRO Orange Concentration (See Note 5 and Fig. 4)
3.2 Protein Stability Assay
3.3 Nucleotide Binding Assay
4 Notes
References
Chapter 16: In Vitro Assembly of a Fully Reconstituted Yeast Translation System for Studies of Initiation and Elongation Phase…
1 Introduction
2 Materials
2.1 Media
2.2 Antibiotics, DNase, and IPTG
2.3 Buffers
2.4 Strains and Plasmids
3 Methods
3.1 Purification of Ribosomal Subunits from S. Cerevisiae
3.2 Purification of Initiation Factors
3.3 In Vitro Hypusination of eIF5A
3.3.1 Purification of eIF5A Hypusination Enzymes Deoxyhypusine Synthase (Dys1) and Deoxyhypusine Hydroxylase (Lia1) (See Note …
3.3.2 In Vitro Hypusination
3.4 RNA Synthesis and Purification
3.5 Nucleotide Binding and Dissociation Assay for eIF5B-397C and eEF1A
3.6 eIF2-GTP-Met-tRNAiMet Ternary Complex Formation Assay
3.7 80S Initiation Complex Formation
3.7.1 Method 1: Size-Exclusion Chromatography
3.7.2 Method 2: Sucrose Cushion Centrifugation
3.8 Peptide Bond Formation Assay
4 Notes
References
Index

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Tags: Karl Dieter Entian, Ribosome Biogenesis