HeLa cytoplasmic extracts contain both 3¢±5¢ and 5¢±3¢ exonuclease activities that may play important roles in mRNA decay. Using an in vitro RNA deadenylation/decay assay, mRNA decay intermediates were trapped using phosphothioate-modi®ed RNAs. These data indicate that 3¢±5¢ exonucleolytic decay is the major pathway of RNA degradation following deadenylation in HeLa cytoplasmic extracts. Immuno-depletion using antibodies speci®c for the exosomal protein PM-Scl75 demonstrated that the human exosome complex is required for ef®cient 3¢±5¢ exonucleolytic decay. Furthermore, 3¢±5¢ exonucleolytic decay was stimulated dramatically by AU-rich instability elements (AREs), implicating a role for the exosome in the regulation of mRNA turnover. Finally, PM-Scl75 protein was found to interact speci®cally with AREs. These data suggest that the interaction between the exosome and AREs plays a key role in regulating the ef®ciency of ARE-containing mRNA turnover.

We have used an in vitro system that reproduces in vivo aspects of mRNA turnover to elucidate mechanisms of deadenylation. DAN, the major enzyme responsible for poly(A) tail shortening in vitro, specifically interacts with the 5' cap structure of RNA substrates, and this interaction is greatly stimulated by a poly(A) tail. Several observations suggest that cap-DAN interactions are functionally important for the networking between regulated mRNA stability and translation. First, uncapped RNA substrates are inefficiently deadenylated. Second, a stem-loop structure in the 5' UTR dramatically reduces deadenylation by interfering with cap-DAN interactions. Third, the addition of cap binding protein eIF4E inhibits deadenylation in vitro. These data provide insights into the early steps of substrate recognition that target an mRNA for degradation.

While decapping plays a major role in mRNA turnover in yeast, biochemical evidence for a similar activity in mammalian cells has been elusive. We have now identi®ed a decapping activity in HeLa cytoplasmic extracts that releases 7me GDP from capped transcripts. Decapping is activated in extracts by the addition of 7me GpppG, which speci®cally sequesters cap-binding proteins such as eIF4E and the deadenylase DAN/PARN. Similar to in vivo observations, the presence of a poly(A) tail represses decapping of RNAs in vitro in a poly(A)-binding protein-dependent fashion. AU-rich elements (AREs), which act as regulators of mRNA stability in vivo, are potent stimulators of decapping in vitro. The stimulation of decapping by AREs requires sequence-speci®c ARE-binding proteins. These data suggest that cap recognition and decapping play key roles in mediating mRNA turnover in mammalian cells.

Three types of exonucleases contribute to the turnover of messenger RNAs in eukaryotic cells: (1) general 3'-to-5' exonucleases, (2) poly(A)-specific 3'-to-5' exonucleases, and (3) 5'-to-3' exonucleases. All three of these activities can be detected in cytoplasmic extracts from a variety of eukaryotic cells. In this chapter, we describe the preparation and use of HeLa cytoplasmic S100 extracts to study these three distinct exonuclease activities. Also included is an immunodepletion protocol that can be used to identify the enzyme responsible for a given activity. These protocols can be easily expanded to the study of trans-acting factors, cis-acting RNA sequence elements, and the interplay of components involved in RNA turnover in various mammalian cell types.

Eighteen new meroterpene derivatives, dichrostachines A-R (1-18), have been isolated from the root and stem barks of Dichrostachys cinerea, and their structures determined by spectroscopic means and molecular modeling. From a biosynthetic standpoint these compounds arise from a Diels-Alder reaction between a labdane diene of the raimonol type and a flavonoid B-ring-derived quinone. The hypothesis was tested by the partial synthesis of similar compounds by simply mixing methyl communate and a synthetic flavonoid quinone. The hemisynthetic compounds were shown by NMR to have configurations different from those of the natural products, thus allowing a refinement of the biosynthesis hypothesis. Most of the compounds were assayed for their ability to inhibit the enzyme protein farnesyl transferase. The most active compounds exhibited IC50 and cytotoxicity values in the 1 microM range.