So how many and what kind of mRNAs are part of Smaug’s treasure? Microarray analyses have shown that at least 20% of all maternal mRNAs are substrates of the maternal degradation pathway [2, 4]. Surprisingly, Smaug is required for the degradation of two-thirds of those, a minimum of 700, and presumably more than 1,000, mRNAs [2]. In their study, Chen et al. [1] examined the mRNAs translationally repressed by Smaug. For this purpose, they isolated polysome-associated mRNA from smg and wild-type control embryos and analyzed them by microarrays. The experiments resulted in a high-confidence set of 342 mRNAs that were more strongly polysome-associated in smg mutants, implying their Smaug-dependent repression in the wild-type. Using a statistical analysis, the authors extrapolated that as many as 3,000 transcripts, about one-half of the total number of mRNAs detectable in the early embryo, may be under translational control by Smaug. However, the two well-known Smaug targets, Hsp83 and nos, were not among them. This was not unexpected: Hsp83 RNA is destabilized but not repressed by Smaug, and nos mRNA has been reported (and was confirmed in this study) to be associated with polysomes, even though translation products are not detectable. This is an important caveat, showing that the presence of an mRNA in polysomal fractions does not exclude regulation by Smaug.

How many of the mRNAs regulated by Smaug are direct targets? Using immunoprecipitation of the protein followed by microarray analysis of associated RNA (RIP-chip), Chen et al. identified transcripts of 339 genes that are bound by Smaug. By means of a recently developed computational method, they then scanned the Smaug-bound RNAs and the high-confidence set of 342 translationally repressed RNAs for the presence of potential SREs, stem-loop structures with the loop sequence CNGGN 0-4 . Both in the bound and in the regulated RNAs, SREs were predicted with a 10-fold higher probability than in non-bound and non-regulated RNAs, respectively. In addition, the selected RNAs contained variant SRE sequences with probabilities matching the binding specificity of Smaug determined in earlier biochemical experiments: high-affinity sites were more enriched than low-affinity sites. These results come as no surprise with regard to the Smaug-bound RNAs; they merely support the reliability of their identification. However, a similar degree of enrichment of the SREs in the translationally repressed RNAs suggests that a large fraction at least of the high-confidence RNAs are direct targets of Smaug. By analyzing data from one of their previous studies [2], the authors also found SREs to be strongly enriched in mRNAs degraded in a Smaug-dependent manner, again suggesting a direct role for Smaug. Performing pairwise comparisons of RNAs bound by Smaug, repressed by Smaug (directly or indirectly) and destabilized by Smaug (directly or indirectly), Chen et al. found high degrees of overlap: two-thirds of the Smaug-bound RNAs were also destabilized by the protein, and three-quarters of the binders were also translationally repressed. Similarly, the destabilized and repressed RNAs overlapped to a large extent.

What about those RNAs that are destabilized or repressed but were not identified as Smaug ligands? These could be regulated indirectly by Smaug or they could be false-negatives in the RIP-chip experiments. From a significant enrichment of SREs in these classes of RNAs, Chen et al. concluded that a large fraction of the regulated RNAs are in fact direct targets of Smaug that escaped detection by RIP-chip.

As the number of Smaug-regulated mRNAs is large, they code for proteins involved in many aspects of biology. Messenger RNAs localized to the posterior pole were prominent among the Smaug targets, as were those encoding proteins involved in the regulation of DNA replication and transcription. More unexpectedly, the list of targets predicts regulatory effects of Smaug on protein folding and proteasome-dependent protein degradation, lipid droplets and even basic energy metabolism. With regard to metabolism, the majority of glycolytic enzymes were identified as potential Smaug targets, and enzyme assays confirmed a modest increase in hexokinase and phosphofructokinase activity in smg mutants.