1 THE REGULATION OF GENE EXPRESSION IN PLANTS AND ANIMALS ...... 1
Robert E. Farrell, Jr.
1.1 OVERVIEW OF EUKARYOTIC TRANSCRIPTION .................... 1
1.1.1 Regulation of Gene Expression .................... 1
1.1.2 Nature of Transcription .......................... 3
1.1.3 Transcription Factors and Promoter Elements ...... 7
1.1.4 Chromosomal Structure Influences Gene
Expression ...................................... 11
1.1.5 Extranuclear Transcriptionally Active
Compartments: Mitochondria and Chloroplasts ..... 12
1.1.6 Types of Nuclear Transcripts Produced ........... 14
1.2 TRANSLATION OF NUCLEAR TRANSCRIPTS ..................... 16
1.2.1 mRNA Sequence and Structure Affect
Translation ..................................... 17
1.2.2 Non-Canonical Initiation of Translation ......... 21
1.2.3 Role of Secondary mRNA Structure on
Translational Control ........................... 21
1.3 MAINSTREAM MOLECULAR TECHNIQUES TO STUDY RNA AS
A PARAMETER OF GENE EXPRESSION ......................... 22
1.3.1 Non-PCR Methods: Northern Analysis, Nuclease
Protection, and Nuclear Runoff Assay ............ 23
1.3.2 PCR-Based Methods: 5' RACE (Rapid
Amplification of cDNA Ends) ..................... 25
1.3.3 In Silico Tools ................................. 28
1.3.4 In Vitro Translation and Western Analysis ....... 30
1.3.5 Implications for Proteomics ..................... 31
1.4 SUMMARY ................................................ 32
REFERENCES .................................................. 34
2 MULTIPLE TRANSCRIPT INITIATION AS A MECHANISM FOR
REGULATING GENE EXPRESSION .................................. 39
Robert E. Farrell, Jr. and Carole L. Bassett
2.1 NUCLEAR GENE TRANSCRIPTION - AN OVERVIEW ............... 39
2.1.1 Initiation of Transcription: Transcription
Factors and Promoter Elements ................... 41
2.1.2 Transcription of Cytoplasmic Genomes ............ 43
2.1.3 Organellar vs Cytoplasmic mRNAs ................. 43
2.2 THE ORIGINS Of MULTIPLE TRANSCRIPTS .................... 44
2.2.1 Multiple Promoters .............................. 44
2.2.2 Transcription Start Sites in Introns ............ 45
2.2.3 Multiple TATA Boxes in a Single Promoter ........ 46
2.2.4 How Alternative TSSs Influence Gene
Expression ...................................... 47
2.3 BICISTRONIC mRNAs ...................................... 52
2.3.1 Moncistronic vs. Polycistronic mRNA ............. 52
2.3.2 Classical Bicistronic mRNA(s) in Plants ......... 55
2.4 CONCLUSION ............................................. 58
REFERENCES .................................................. 59
3 ALTERNATIVE PROCESSING AS A MECHANISM FOR REGULATING GENE
EXPRESSION .................................................. 67
Eliezer S. Louzada
3.1 INTRODUCTION ........................................... 67
3.2 REGULATION OF ALTERNATIVE SPLICING ..................... 68
3.2.1 Splice Site Recognition ......................... 68
3.2.2 Factors Affecting Canonical and Alternative
Splicing ........................................ 71
3.3 MODE OF ACTION OF ALTERNATIVE SPLICING ................. 77
3.3.1 Exon Skipping ................................... 78
3.3.2 Intron Retention ................................ 81
3.3.3 Cryptic Introns ................................. 83
3.4 FUNCTIONAL SIGNIFICANCE OF ALTERNATIVE SPLICING ........ 84
3.4.1 Nonsense-Mediated mRNA Decay .................... 84
3.4.2 Control of Gene Expression ...................... 86
3.4.3 Alternative Splicing and Stress ................. 88
3.5 CONCLUSION AND PROSPECTUS .............................. 89
REFERENCES .................................................. 91
4 MESSENGER RNA 3'-END FORMATION AND THE REGULATION OF GENE
EXPRESSION ................................................. 101
Arthur G. Hunt
4.1 INTRODUCTION AND AN OVERVIEW OF POLYADENYLATION ....... 101
4.2 POLYMORPHISM IN POLYADENYLATION SITES IN PLANTS ....... 105
4.2.1 Regulation via mRNA 3' end Processing .......... 105
4.2.2 The Scope of Alternative Polyadenylation in
Plants ......................................... 108
4.3 REGULATION OF POLYADENYLATION IN PLANTS ............... 110
4.3.1 Recent Developments Regarding the Nature
of Polyadenylation Signals in Plants ........... 110
4.3.2 Polyadenylation Signals and Alternative 3'
end Processing ................................. 112
4.3.3 Involvement of Proteins Apart from
Polyadenylation Factor Subunits in 3' end
Processing ..................................... 114
4.3.4 Linking Polyadenylation to Environmental and
Developmental Cues ............................. 115
REFERENCES ................................................. 117
5 AN OVERVIEW OF SMALL RNAs .................................. 123
Jean-Michel Hily and Zongrang Liu
5.1 SMALL RNAs: TARGETS AND MECHANISMS .................... 123
5.1.1 Distinguishing Between the Small RNAs: siRNA,
miRNA, and Other Small RNAs .................... 124
5.1.2 Two Distinct Stages of RNAi: Initiator and
Effector Phases ................................ 126
5.1.3 Operational Modes and Functions ................ 129
5.1.4 Amplification of the Silencing Triggers ........ 133
5.1.5 A Natural Defense Mechanism .................... 133
5.2 USING RNAi TECHNOLOGY AS A MOLECULAR TOOL ............. 134
5.2.1 Methods of Induction of Gene Silencing ......... 135
5.2.2 Functional Genomic Tools to Understand
Essential Regulation of Key Developmental
Processes ...................................... 136
5.2.3 Improvement of Plant Characteristics ........... 137
5.3 CONCLUSION ............................................ 139
REFERENCES ................................................. 141
6. CONTROL OF GENE EXPRESSION BY mRNA TRANSPORT AND
TURNOVER ................................................... 148
Carole L. Bassett
6.1 INTRODUCTION .......................................... 148
6.2 mRNA TRANSPORT AND LOCALIZATION ....................... 148
6.2.1 mRNA Transport ................................. 149
6.2.2 mRNA Localization .............................. 152
6.2.3 RNA Granules ................................... 153
6.2.4 Nuclear Compartments ........................... 159
6.3 mRNA BINDING FACTORS .................................. 161
6.3.1. mRNPs .......................................... 161
6.4 mRNA TURNOVER ......................................... 167
6.4.1 General mRNA Decay ............................. 168
6.4.2 mRNA Surveillance .............................. 169
6.5 SUMMARY AND PROSPECTUS ................................ 174
REFERENCES ................................................. 175
SUBJECT INDEX ................................................. 189
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