Smaug triggers destabilization and localization of specific maternal transcripts through recruitment of the CCR4/POP2/NOT deadenylase. In contrast, Smaug-mediated translational repression is accomplished via an indirect interaction between Smaug and eIF4E, a component of the basic translation machinery. Thus, Smaug is a multifunctional posttranscriptional regulator that employs distinct mechanisms to repress translation and to induce degradation of target transcripts.

SMAUG (SMG) is an RNA-binding protein that functions as a key component of a transcript degradationpathway that eliminates maternal mRNAs in the bulk cytoplasm of activated Drosophila melanogaster eggs. We previously showed that SMG destabilizes maternal Hsp83 mRNA by recruiting the CCR4-NOT deadenylase to trigger decay; however, the cis-acting elements through which this was accomplished were unknown. Here we show that Hsp83 transcript degradation is regulated by a major element, the Hsp83 mRNA instability element (HIE), which maps to a 615-nucleotide region of the open reading frame (ORF). The HIE is sufficient for association of a transgenic mRNA with SMG protein as well as for SMG-dependent destabilization. Although the Hsp83 mRNA is translated in the early embryo, we show that translation of the mRNA is not necessary for destabilization; indeed, the HIE functions even when located in an mRNA's 3 untranslated region. The Hsp83 mRNA contains eight predicted SMG recognition elements (SREs); all map to the ORF, and six reside within the HIE. Mutation of a single amino acid residue that is essential for SMG's interaction with SREs stabilizes endogenous Hsp83 transcripts. Furthermore, simultaneous mutation of all eight predicted SREs also results in transcript stabilization. A plausible model is that the multiple, widely distributed SREs in the ORF enable some SMG molecules to remain bound to the mRNA despite ribosome transit through any individual SRE. Thus, SMG can recruit the CCR4-NOT deadenylase to trigger Hsp83 mRNA degradation despite the fact that it is being translated.

Modulating the efficiency of translation plays an important role in a wide variety of cellular processes and is often mediated by trans-acting factors that interact with cis-acting sequences within the mRNA. Here we show that a cis-acting element, the Hsp83 degradation element (HDE), within the 3-untranslated region of the Drosophila Hsp83 mRNA functions as a translational enhancer. We show that this element is bound by a multiprotein complex, and we identify components using a novel affinity-based method called tandem RNA affinity purification tagging. Three proteins (DDP1, Hrp48, and poly(A)-binding protein) are components of the HDE-binding complex and function in translational enhancement. Our data support a model whereby the HDE is composed of several cis-acting subelements that represent binding sites for trans-acting factors, and the combined action of these trans-acting factors underlies the ability of the HDE to stimulate translation.Regulated translation plays an essential role in a wide variety of cellular processes. Although translational regulation is likely to function in virtually all eukaryotic cell types, these controls are particularly important in cells where transcriptional regulation is not an option. For example, maturation of mammalian red blood cells occurs after the nucleus is extruded and thus is driven by previously synthesized mRNAs. Similarly, in early metazoan embryos, the zygotic genome is transcriptionally silent, and maternally deposited mRNAs control early development. Translational regulation is also very important in large cells, such as neurons, where correct spatial and temporal expression of proteins cannot be achieved through transcriptional controls alone.Regulation of specific transcripts is often mediated by cisacting elements within the 5Ј-or 3Ј-untranslated region (UTR) 4 of an mRNA (1). These elements can act as binding sites for trans-acting factors that either directly or indirectly contact the translational machinery. Some of the best characterized mechanisms serve to repress protein expression, but mechanisms that stimulate protein production also exist. In principle, these positively acting events can be divided into two different classes. The first acts on transcripts that are translationally repressed. Translational stimulation is achieved by blocking the repressive mechanism (i.e. enhancement results from relief of repression). The second class of stimulatory events acts on mRNAs that are not repressed. In these cases, an mRNA is better able to recruit the basic translation machinery and is, therefore, expressed at a higher level. This latter type of mechanism is likely to be particularly important when a component of the translation machinery is limiting and, consequently, transcripts must compete for access to the translational apparatus.Many viral RNAs contain elements that aid in preferential expression in infected cells. For example, the 5Ј-UTR of the tobacco mosaic virus RNA contains a cis-acting element, ⍀, that is bound by Hsp101, which in turn recrui...