Chapter 13. Identifying substrates of mRNA decay factors by a combined RNA interference and DNA microarray approach.
The complex control of gene expression has many layers, and the modulation of posttranscriptional events receives more and more attention as a focus of research. In this respect, regulation of mRNA turnover is important, as the differential longevity of an mRNA enables a cell to rapidly alter the abundance of a protein in response to intra- and extracellular signals. While the list of factors known to catalyze or regulate mRNA decay is steadily increasing, the substrate specificities of most of these factors, as well as their precise roles in the degradation of individual mRNAs, have remained elusive. One approach for determining the substrate repertoire of a particular mRNA decay factor involves a genome-wide DNA microarray analysis of mRNAs that accumulate in cells in which the abundance of the respective factor has been reduced by RNA interference. Using the knockdown of the nonsense-mediated mRNA decay factor human UPF2 as a model system, this chapter provides a detailed protocol of how to reduce the abundance of an mRNA decay factor by small interfering RNAs and to determine differential mRNA profiles by a subsequent DNA microarray analysis. Our combined RNA interference/DNA microarray approach, as well as all experimental protocols, can, however, be easily adapted to any mRNA decay factor of interest.
Wittmann
Jürgen
J
Division of Molecular Immunology, Department of Internal Medicine III, Nikolaus Fiebiger Center, University of Erlangen-Nürnberg, Erlangen, Germany.
Jäck
Hans-Martin
HM
D001483:Base Sequence; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference / Q000502:physiology*; D020871:RNA Stability / Q000235:genetics*; D016601:RNA-Binding Proteins; D014157:Transcription Factors / Q000235:genetics
D016601:RNA-Binding Proteins; D014157:Transcription Factors; C468286:UPF2 protein, human