Rapid ATP-dependent Deadenylation of nanos mRNA in a Cell-free System from Drosophila Embryos

Abstract

Shortening of the poly(A) tail (deadenylation) is the first and often rate-limiting step in the degradation pathway of most eukaryotic mRNAs and is also used as a means of translational repression, in particular in early embryonic development. The nanos mRNA is translationally repressed by the protein Smaug in Drosophila embryos. The RNA has a short poly(A) tail at steady state and decays gradually during the first 2-3 h of development. Smaug has recently also been implicated in mRNA deadenylation. To study the mechanism of sequence-dependent deadenylation, we have developed a cell-free system from Drosophila embryos that displays rapid deadenylation of nanos mRNA. The Smaug response elements contained in the nanos 3-untranslated region are necessary and sufficient to induce deadenylation; thus, Smaug is likely to be involved. Unexpectedly, deadenylation requires the presence of an ATP regenerating system. The activity can be pelleted by ultracentrifugation, and both the Smaug protein and the CCR4⅐NOT complex, a known deadenylase, are enriched in the active fraction. The same extracts show pronounced translational repression mediated by the Smaug response elements. RNAs lacking a poly(A) tail are poorly translated in the extract; therefore, SRE-dependent deadenylation contributes to translational repression. However, repression is strong even with RNAs either bearing a poly(A) tract that cannot be removed or lacking poly(A) altogether; thus, an additional aspect of translational repression functions independently of deadenylation.

Subunits of the Drosophila CCR4-NOT complex and their roles in mRNA deadenylation

“…These results confirmed the specificity of the antibodies and suggested that the two polypeptides were isoforms of NOT1. Analysis with antibodies directed against the other anticipated subunits of the CCR4-NOT complex Jeske et al 2006) revealed a depletion of CAF1, CCR4, NOT1, NOT2, and NOT3 in the supernatant of the immunoprecipitation. The same polypeptides were enriched in the peptide eluate (Fig.…”

“…Brain Tumor interacts with the regulatory protein 4EHP (Cho et al 2006), which was also present in the NOT1 precipitate, although its representation with a single peptide was below the threshold set for protein identification. Bicaudal is involved in post-transcriptional regulation of the nanos RNA, which is a substrate for the CCR4-NOT complex (Markesich et al 2000;Jeske et al 2006;Zaessinger et al 2006). …”

“…Alternatively, miRNAs can be responsible for the recruitment of the CCR4-NOT complex to specific mRNAs (Behm-Ansmant et al 2006;Fabian et al 2009). ATP dependence of a cell-free deadenylation system derived from Drosophila embryos suggests the involvement of additional factors (Jeske et al 2006). The CCR4-NOT complex may also play a role in transcriptional regulation (Collart 2003;Denis and Chen 2003;Collart and Timmers 2004).…”

Mutation of mouse Samd4 causes leanness, myopathy, uncoupled mitochondrial respiration, and dysregulated mTORC1 signaling

“…In Drosophila, Smaug inhibits translation by blocking the formation of a competent translation initiation complex (9,23), by promoting deadenylation of target mRNAs leading to their destabilization and degradation (24)(25)(26), and by micro-RNA-independent recruitment of Argonaute 1 (Ago1) to target mRNAs (27). RNA immunoprecipitation combined with microarray analysis demonstrated that Smaug binds directly to numerous embryonic fly RNAs, including those encoding glycolytic enzymes, and components of the proteasome regulatory particle, the TRiC/CCT chaperonin, and lipid droplets (28).…”

Trading translation with RNA-binding proteins

“…Cup is recruited to oskar and nanos mRNAs by the 39 UTR binding proteins Bruno and Smaug, respectively. As for hunchback, additional mechanisms contribute to translational repression of these transcripts, including Bruno-driven mRNA oligomerization and Smaug-dependent deadenylation (Chekulaeva et al 2006;Jeske et al 2006;Zaessinger et al 2006). The examples of hunchback and nanos mRNAs illustrate that deadenylation often cooperates with mechanisms of translational control FIGURE 1.…”