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

Mandy Jeske,Dorian Freudenreich,Claudia Temme,Elmar Wahle,Sylke Meyer

doi:10.1074/jbc.m604802200

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 CCR4.NOT 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.

Highlights

Different mRNAs decay at different rates, which are largely determined by the rates of deadenylation; stable RNAs are deadenylated slowly, and unstable RNAs are deadenylated rap
The system that we describe here should be amenable to biochemical resolution and reconstitution and offers the promise of combining biochemistry with Drosophila genetics
Because the CCR41⁄7NOT complex is implicated in the deadenylation not just of nos but of many RNAs in all eukaryotes, the results should be relevant for sequence-dependent rapid deadenylation in general

Summary

The abbreviations used are

3Ј-UTR, 3Ј untranslated region; PARN, poly(A)specific ribonuclease; SRE, Smaug response element; ATP␥S, adenosine 5Ј-O-(thiotriphosphate); AMPPNP, adenosine 5Ј-(␤,␥-imino)triphosphate; AMPPCP, adenosine 5Ј-(␤,␥-methylene)triphosphate; nt, nucleotide(s). The large majority of the nos RNA is distributed uniformly throughout the embryo [52, 53] Translation of this unlocalized RNA is repressed by the protein Smaug, which binds two Smaug response elements (SREs) in the proximal part of the nos 3Ј-UTR (54 –57). Several lines of evidence indicated that the regulation of nos RNA might involve poly(A) tail shortening induced by Smaug; a Saccharomyces cerevisiae homolog of Smaug, Vts1p, binds SREs and leads to accelerated decay of SRE-containing reporter RNAs, which requires the deadenylase subunit Ccr4p [60]. Translation is strongly enhanced by the poly(A) tail in the embryo extract, so SRE-dependent deadenylation represses translation.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION