Measuring mRNA stability during early Drosophila embryogenesis.
Maternal mRNAs play a major role in directing early Drosophila melanogaster development, and thus, precise posttranscriptional regulation of these messages is imperative for normal embryogenesis. Although initially abundant on egg deposition, a subset of these maternal mRNAs is targeted for destruction during the first 2 to 3 h of embryogenesis. In this chapter, we describe molecular methods to determine the kinetics and mechanisms of maternal mRNA decay in the early D. melanogaster embryo. We show how both unfertilized eggs and fertilized embryos can be used to identify maternal mRNAs destined for degradation, to explain changes in decay kinetics over time, and to uncover the molecular mechanisms of targeted maternal mRNA turnover. In the first section, we explore the methods and outcomes of measuring decay on a "gene-by-gene" basis, which involves examination of a small number of transcripts by Northern blotting, RNA dot blotting, and real-time RT-PCR. In the second section, we provide a comprehensive examination of the applications of microarray technology to study global changes in maternal mRNA decay during early development. Genome-wide surveys of maternal mRNA turnover provide a wealth of information regarding the magnitude, temporal regulation, and genetic control of maternal mRNA turnover. Methods that permit the collection and analysis of highly reproducible and statistically robust data in this developmental system are discussed.
Semotok
Jennifer L
JL
Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Ontario, Canada.
Westwood
J Timothy
JT
Goldman
Aaron L
AL
Cooperstock
Ramona L
RL
Lipshitz
Howard D
HD
D000818:Animals; D004331:Drosophila melanogaster / Q000196:embryology* / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000196:embryology* / Q000378:metabolism*; D047108:Embryonic Development; D018507:Gene Expression Regulation, Developmental; D049750:Genome, Insect / Q000235:genetics; D020871:RNA Stability; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D012333:RNA, Messenger