Nelson Jonathan O, Metzstein Mark M
Department of Human Genetics, University of Utah, Salt Lake City, UT, USA.
Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.
Methods Mol Biol. 2018;1720:205-211. doi: 10.1007/978-1-4939-7540-2_15.
Transcriptome analysis provides a snapshot of cellular gene expression and is used to determine how cells and organisms respond to genetic or environmental changes. Identifying the transcripts whose expression levels are regulated directly by the manipulation being examined from those whose expression changes as a secondary cause from the primary changes requires additional analyses. Here we present a technique used to distinguish direct targets of the nonsense-mediated mRNA decay (NMD) pathway in Drosophila from secondary gene expression effects caused by loss of this pathway. This technique uses pulsed reexpression of an essential NMD gene in Drosophila lacking this NMD factor, followed by analysis of the transcriptome over time. In this way, RNAs with a rapid reduction in expression upon reactivation of NMD activity, corresponding to primary NMD targets, can be identified. This technique could potentially be modified to identify direct targets of other mRNA decay mechanisms in Drosophila or other organisms.
转录组分析提供了细胞基因表达的一个快照，并用于确定细胞和生物体如何响应遗传或环境变化。从那些因主要变化而作为次要原因发生表达变化的转录本中，识别出其表达水平直接受所研究操作调控的转录本，需要进行额外的分析。在这里，我们展示了一种用于区分果蝇中无义介导的mRNA降解（NMD）途径的直接靶标与该途径缺失所导致的次要基因表达效应的技术。该技术利用在缺乏这种NMD因子的果蝇中对一个必需的NMD基因进行脉冲式重新表达，随后对转录组随时间进行分析。通过这种方式，可以识别出在NMD活性重新激活时表达迅速降低的RNA，这些RNA对应于主要的NMD靶标。该技术有可能被改进，以识别果蝇或其他生物体中其他mRNA降解机制的直接靶标。
