Drosophila 5′ → 3′-exoribonuclease Pacman

This chapter concentrates on the methods used to express a Drosophila recombinat 5? ? 3?-exoribonuclease, purify the protein, and analyze its activity in vitro. Analysis of early development in Drosophila has shown that RNA localization, control of translation, and mRNA stability are intimately linked. Generally, translational repression leads to degradation of an RNA, and failure of an RNA to localize correctly also leads to its degradation. Work on the yeast Saccharomyces cerevisiae has identified many ribonucleases and associated factors that control mRNA decay, RNA splicing, and rRNA processing. In yeast, it has been shown that 3? ? 5? degradation/processing of RNA requires the exosome, which is a complex of at least 10 proteins. Degradation of RNA in a 5? ? 3? direction occurs by initial decapping of the mRNA, followed by 5? ? 3? degradation of the RNA by Xrn1p. The two decapping proteins (Dcp1p and Dcp2p) and Xrnlp have been shown to be complexed to seven Lsm proteins, which are likely to form a ring encircling the RNA. To understand the role of RNA stability in development, a number of approaches can be used. Once the genes encoding particular ribonucleases or associated factors have been identified, then expression of the RNA during development can be determined. These techniques have been used to show that the 5? ? 3?-exoribonuclease Pacman is differentially expressed during development.