The Role of Smaug, an RNA-binding protein, in mRNA translation and stability in the early Drosophila embryo


Differential gene expression is a critical part of early metazoan development, and cells have many mechanisms for spatial and temporal control of gene expression. Genes are first transcribed into mRNA in the cell nucleus and then translated into functional proteins in the cytoplasm. One mechanism by which the cell can control gene expression is through post-transcriptional regulation, or more simply, control of gene expression at the RNA level. Post-transcriptional regulation can occur through mechanisms such as RNA capping, splicing, polyadenylation, or other methods that affect mRNA stability and degradation.

During early metazoan development, post-transcriptional regulation of maternal RNA is important in the Maternal-to-Zygotic Transition (MZT). Transcripts from the mother’s genome, which are deposited into the developing oocyte, regulate protein synthesis via mRNA translation, stabilization, and localization. Activation of the MZT requires not only activation of the zygotic genome, but also the destruction of much of the maternal RNA in the embryo.1

These maternal transcripts can be regulated by cis-acting elements, which are sequences that serve as binding sites for microRNA (miRNA) or RNA-binding proteins. These bound miRNA and RNA-binding proteins can inhibit translation of the maternal mRNA or can induce degradation. One such RNA-binding protein, Drosophila Smaug, is highly conserved from yeast to humans.2 Smaug is a sequence-specific, multifunctional post-transcriptional regulator that can inhibit translation or promote degradation of target mRNAs in the early Drosophila embryo.

Smaug binds to cis-acting elements of its target mRNA through stem-loop structures, which are known as Smaug recognition elements (SRE’s). These elements include sterile alpha motifs (SAMs), which serve as the direct binding site for Smaug.

Smaug Inhibits nanos Translation

Maternal nanos (nos) mRNA is a crucial morphogen that assists in the development of the anteroposterior axis in early Drosophila embryos. Although nos is distributed throughout the bulk cytoplasm of the embryo, its functional protein is only expressed at the posterior. Smaug binds nos not localized to the posterior and inhibits its translation. The binding of Smaug to nos occurs via two SRE’s located in the nos 3′ UTR.3

One of the mechanisms whereby Smaug can regulate nos translation is via recruitment of Argonaute (Ago) proteins. Usually, Ago proteins will bind to small, non-coding RNAs, such as miRNA, and then attach to the target mRNA via base pairing with the miRNA. Interestingly, Pinder & Smibert found that Ago proteins can bind directly to Smaug without needing any RNAs. The Smaug-Ago protein complex will then go on to repress translation of the target mRNA (Fig. 1).4


Fig 1. Two mechanisms for Argonaute (Ago) recruitment. (a) MiRNA (blue strand) guides Ago to the target mRNA via base pairing of partially complementary sequences. Recruitment of the GW protein leads to translational repression. (b) An RNA-binding protein (RBP), such as Smaug, first binds to the target mRNA and then recruits the Ago-GW protein complex. This also leads to translational repression.5

Another mechanism of nos regulation occurs via the interaction between Smaug and Cup protein. After Smaug binds to the SRE’s in the nos 3′ UTR, it recruits Cup protein. Cup then interacts with another protein, eukaryotic translation initiation factor 4E (eIF4E). Normally, eIF4E would form a complex with eIF4G and recruit the 40S ribosomal subunit of the translational machinery, leading to an increase in translation. However, Cup binding to eIF4E inhibits eIF4G binding and leads to translational repression (Fig. 2).6


Fig 2. Smaug binds to maternal nanos mRNA at the SRE located in the 3’UTR. Cup is recruited and binds to Smaug. Cup also binds eIF4E, which inhibits formation of an eIF4E-eIF4G complex. This leads to translational repression of the transcript.7


Smaug-Dependent Degradation of nanos and Hsp83 mRNA

The poly(A) tail, located on an mRNA’s 3′ terminus, stabilizes the transcript and prevents enzymatic degradation. Loss of this poly(A) tail (i.e. deadenylation) enhances degradation of the transcript. Nos and Hsp83, another well-studied Drosophila maternal mRNA, are destabilized and degraded via Smaug-dependent deadenylation. Degradation of both transcripts occurs in the bulk cytoplasm of the early embryo, but the transcripts are protected at the posterior pole plasm, resulting in localization.

Smaug-nos binding has been discussed previously. Smaug binding to Hsp83 occurs via eight SRE’s located in the open reading frame (ORF).8 Following binding, Smaug recruits the eight-subunit CCR4-NOT deadenlyase complex. Figure 3 shows the composition of the Drosophila CCR4-NOT complex, and Figure 4 shows Smaug-mediated CCR4-NOT binding to the target transcript. CCR4 and POP2 possess exonucleolytic activity that is specific to the 3′ poly(A) tail. These two proteins are therefore responsible for the actual cleavage of the adenine residues.9

General CCR4:NOT

Fig 3. The Drosophila CCR4-NOT complex. (A) The eight subunits and their respective sizes. Rectangles represent structured domains. (B) Interactions between the subunits and the structure of the complex as a whole.10

CCR4:NOT Complex

Fig 4. Smaug-dependent binding of the CCR4-NOT complex to nanos and subsequent deadenylation of the transcript.10











Smaug’s mRNA Targets

Although nos and Hsp83 are well-characterized in their interactions with Smaug, they are not the only targets of the RNA-binding protein. Chen et al. have shown that Smaug may control the translation of more than 3000 mRNAs, and of those, more than 1000 may be degraded in a Smaug-dependent manner.

Genome-wide experiments have shown that more than 1000 maternal transcripts exhibit increased stability in embryos from smaug homozygous-mutant females.2 In order to understand which transcripts interact with Smaug, Chen et al. used RNA co-immunoprecipitation (RIP-ChIP) followed by DNA microarray hybridization. They identified 339 genes whose transcripts were significantly enhanced in Smaug immunoprecipitations (at a high confidence level). In addition, they used polysome gradients and microarrays to determine 342 mRNAs that are translationally repressed by Smaug (again, at a high confidence level). Extrapolation of this data to account for false negatives at lower confidence levels suggests that Smaug controls translation of about 3000 transcripts.  Comparison of those transcripts that are bound to Smaug with those that are translationally repressed and those that are degraded shows that a large fraction are both repressed and degraded by Smaug (Fig. 5).

Smaug Bound:Repressed:Degraded

Fig 5. Comparison of transcripts bound, translationally repressed, and degraded by Smaug. Venn diagrams show overlap between groups, indicating that a large fraction of transcripts are both translationally repressed and degraded by Smaug.



  1. Götze, M., & Wahle, E. (2014). Smaug destroys a huge treasure. Genome Biology, 15, 101.
  2. Tadros, W., Goldman, A., Babak, T., Menzies, F., Vardy, L., Orr-Weaver, T., … Lipshitz, H. (2007). SMAUG Is a Major Regulator of Maternal mRNA Destabilization in Drosophila and Its Translation Is Activated by the PAN GU Kinase. Developmental Cell, 12(1), 143-155.
  3. Chen, L., Dumelie, J., Li, X., Cheng, M., Yang, Z., Laver, J., … Smibert, C. (2014). Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein. Genome Biology, 15, R4.
  4. Pinder, B., & Smibert, C. (2013). MicroRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein. EMBO Reports, 14(1), 80-86.
  5. Meister, G. (2013). Argonaute proteins: Functional insights and emerging roles. Nature Reviews Genetics, 14, 447-459.
  6. Nelson, M., Leidal, A., & Smibert, C. (2003). Drosophila Cup is an eIF4E-binding protein that functions in Smaug-mediated translational repression. The EMBO Journal, 23(1), 150-159.
  7. Vardy, L., & Orr-Weaver, T. (2007). Regulating translation of maternal messages: Multiple repression mechanisms. Trends in Cell Biology, 17(11), 547-554.
  8. Semotok, J., Luo, H., Cooperstock, R., Karaiskakis, A., Vari, H., Smibert, C., & Lipshitz, H. (2008). Drosophila Maternal Hsp83 mRNA Destabilization Is Directed by Multiple SMAUG Recognition Elements in the Open Reading Frame. Molecular and Cellular Biology, 28(22), 6757-6772.
  9. Semotok, J., Cooperstock, R., Pinder, B., Vari, H., Lipshitz, H., & Smibert, C. (2005). Smaug Recruits the CCR4/POP2/NOT Deadenylase Complex to Trigger Maternal Transcript Localization in the Early Drosophila Embryo. Current Biology, 15(4), 284-294.
  10. Temme, C., Simonelig, M., & Wahle, E. (2014). Deadenylation of mRNA by the CCR4–NOT complex in Drosophila: Molecular and developmental aspects. Frontiers in Genetics, 5, 143-143.

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