Elsevier S & T

mRNA Formation and Function

Richter, Joel D.

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Table of contents

Cover
Contentsv
Contributorsxiii
Prefacexvii
Chapter 1. The Tat-TAR RNP, a Master Switch That Regulates HIV-1 Gene Expression1
I. Introduction1
II. Methods4
References21
Chapter 2. HeLa Nuclear Extract: A Modified Protocol25
I. Growth of the HeLa Cells25
II. Extract Preparation26
Reference30
Chapter 3. Functional Analysis of Splicing Factors and Regulators31
I. Introduction31
II. Functional Analysis of General Splicing Factors32
III. Analysis of Regulatory Mechanisms in Pre-mRNA Splicing43
References49
Chapter 4. Identification of Pre-mRNA Splicing Factors and Analysis of RNA–Protein Interaction55
I. Introduction55
II. Pre-mRNA Splicing and the Role of the Polypyrimidine Tract56
III. Analysis of RNA Binding Proteins by UV Cross-Linking58
IV. Analysis of Protein-RNA Interaction by Fluorescence Spectroscopy62
V. Analysis of Pre-mRNA Splicing Complexes and Identification of Associated Factors by UV Cross-Link68
VI. Identification of RNA Binding Proteins by a cDNA Expression Library Screen72
References75
Chapter 5. In Vitro Analysis of Mammalian Cell mRNA 3' Processing79
I. Introduction79
II. General Considerations for the Analysis of mRNA 3' Processing in Vitro80
III. Pre-mRNA Cleavage and Polyadenylation82
IV. 3' Processing Complex Formation85
V. 3' Processing Complex Stability88
VI. Probing 3' Processing Factor–Pre-mRNA Interactions by UV Cross-Linking90
VII. Application of in Vitro Selection to the Analysis of Sequences That Direct 3' Processing93
VIII. Conclusions97
References97
Chapter 6. Rapid Identification and Cloning of Sequence-Specific RNA Binding Proteins99
I. Introduction99
II. Preparation of Extracts100
III. Preparation of RNAs101
IV. Identification of Protein–RNA Complexes102
V. Determination of RNA Sequence Requirements for Protein Binding103
VI. Cloning cDNAs for RNA Binding Proteins by Northwestern Screening105
References108
Chapter 7. Analysis of Polyadenylation Phenotypes in Saccharomyces cerevisiae111
I. Introduction111
II. Poly(A) Tail Analysis112
III. Polyadenylation Site Mapping by RT-PCR117
IV. Preparation of Yeast mRNA 3' End Processing Extracts121
References125
Chapter 8. Poly(A) Polymerase/Cap-Specific 2'-O-Methyltransferase from Vaccinia Virus: Expression, P127
I. Background127
II. Expression and Purification of the Poly(A) Polymerase/ 2'- O-Methyltransferase132
III. Uses of the Poly(A) Polymerase/2'-O-Methyltransferase135
IV. Assays Developed to Examine Protein–Ligand Interactions139
References145
Chapter 9. A Genetic Approach to Mapping Coding Region Determinants o f mRNA Instability in Yeast149
I. Introduction149
II. Experimental Methodology150
III. Experimental Results153
IV. Discussion and Conclusions157
References160
Chapter 10. Identification of the Protein That Interacts with the 3' End of Histone mRNA163
I. Introduction163
II. Detecting Specific Protein–RNA Interactions165
III. In Vitro Processing of Histone Pre-mRNA174
IV. Isolation of Polyribosomes181
V. Isolation and Characterization of RNA Binding Proteins Using the Yeast Three-Hybrid System183
Appendix: Cell Culture188
References192
Chapter 11. Analysis of mRNP Complexes Assembled in Vitro195
I. Complex Formation196
II. Detection of RNP Complexes200
III. Analysis of RNP Complexes Identified by Electrophoretic Mobility Shift Analysis202
References208
Chapter 12. Analysis of RNA Binding Proteins Using in Vitro Genetics211
I. Introduction212
II. Successfully Making Display Phage215
III. Binding of Displayed RNA Binding Proteins to RNA221
IV. Application of in Vitro Genetics to the Study of RNA Binding Proteins224
V. Protocols227
References234
Chapter 13. Interactions of Proteins with Specific Sequences in RNA237
I. Introduction237
II. Selection of Specific Sequences from Random RNA Libraries by RNA Binding Proteins as a First Ste240
III. Binding of Protein to Sequences in Cellular RNAs247
IV. Determination of Minimal Binding Sequences within RNA Ligands250
V. Selection of RNAs from 3' UTR Libraries257
VI. Summary259
References259
Chapter 14. A Rapid Genetic Method for the Study of RNA Binding Proteins263
I. Introduction263
II. Background264
III. A Quantitative Model for Translation Repression by Heterologous RNA-BPs265
IV. Methods267
V. Strategies and Applications274
VI. Troubleshooting280
VII. Application of the Genetic Methods to Rev281
VIII. Conclusions282
References283
Chapter 15. Footprinting RNA–Protein Complexes with Hydroxyl Radicals285
I. Introduction286
II. Preparing an RNA Template for Hydroxyl Radical Footprinting288
III. Binding and Cleavage Reactions292
IV. Footprinting Data293
V. Concluding Remarks299
References300
Chapter 16. Analysis of Ribosome Loading onto mRNA Species: Implications for Translational Control305
I. Polysome Profiles and Translational Control305
II. Extraction and Display of Polysomes307
III. RNA Analysis by Northern Blots312
IV. Analysis by RNase Protection315
References320
Chapter 17. Analysis of Picornavirus Internal Ribosome Entry Site Function in Vivo323
I. Introduction323
II. Cap-Dependent and Cap-Independent Translation324
III. Characteristics of Picornavirus IRES Elements325
IV. Characteristics of the Vaccinia/T7 RNA Polymerase Expression System327
V. Analysis of Picornavirus IRES Function in Vivo328
VI. Distinguishing between Cap-Dependent and IRES- Directed Translation334
VII. Complementation of Defective IRES Elements by Co- Expression of the wt IRES in Trans335
References337
Chapter 18. RNA Traffic and Localization Reported by Fluorescent Molecular Cytochemistry in Living C341
I. Introduction341
II. Fluorescent Labeling of RNA342
III. Cell Culture, Microinjection, and Digital Imaging Processing344
IV. Examples350
V. What Is the Biological Authenticity of Fluorescent RNA?351
VI. Technical Problems and Their Resolution353
VII. Integrating Fluorescent RNA Cytochemistry with Other Methods354
VIII. What Does Fluorescent RNA Cytochemistry Actually Measure?355
IX. New Frontier Methods356
X. Closing Remarks356
References357
Chapter 19. Detection of mRNA in Situ: Techniques for Studying Gene Expression in Drosophila melanog361
I. Introduction361
II. Whole-Mount in Situ Hybridization to mRNA with DIG-Labeled Probes362
III. Uses of the Whole-Mount Procedure365
IV. Detection of Multiple Gene Products367
V. Subcellular Localization of wingless mRNA369
References369
Chapter 20. Differential Display Protocol That Preferentially Identifies mRNAs of Moderate to Low Ab371
I. Introduction371
II. Experimental Procedures373
III. Confirmation of Differential Expression379
IV. Concluding Remarks381
References382
Index385
Color Plate Sections397

Book details

Vendor Elsevier S & T
SKU 9780125875455
ISBN-13 9780080537047
Author Richter, Joel D.
Category Science
Subject Genetics & Genomics

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mRNA Formation and Function presents a compendium of techniques geared exclusively toward the understanding of RNA metabolism. It will be particularly useful because a number of different organisms and systems are employed.

Key Features
* Isolation and characterization of specific RNA binding proteins
* RNA metabolism and associated regulatory proteins
* RNA detection and localization
* A genetic approach to RNA function

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