Maternal mRNA deadenylation and decay by the piRNA pathway in the early Drosophila embryo

Piwi-associated RNAs (piRNAs), a specific class of 24- to 30-nucleotide-long RNAs produced by the Piwi-type of Argonaute proteins have a specific germline function in repressing transposable elements. This repression is thought to involve heterochromatin formation, transcriptional and posttranscriptional silencing^1–6. The piRNA pathway has other essential functions in germline stem cell maintenance⁷ and in maintaining germline DNA integrity^8–10. Here, we uncover an unexpected function of the piRNA pathway in the decay of maternal mRNAs and in translational repression in the early embryo. A subset of maternal mRNAs is degraded in the embryo, at the maternal to zygotic transition. In Drosophila, maternal mRNA degradation depends on the RNA binding protein Smaug (Smg) and the deadenylase CCR4^11–13, as well as the zygotic expression of a microRNA (miRNA) cluster¹⁴. Using mRNA encoding the embryonic posterior morphogen Nanos (Nos) as a paradigm to study maternal mRNA decay, we found that CCR4-mediated deadenylation of nos depends on components of the piRNA pathway including piRNAs complementary to a specific region in nos 3′-UTR. Reduced deadenylation when piRNA-induced regulation is impaired, correlates with nos mRNA stabilization and translational derepression in the embryo, resulting in head development defects. Aubergine (Aub), one of the Argonaute proteins in the piRNA pathway, is present in a complex with Smg, CCR4, nos mRNA and piRNAs that target nos 3′-UTR, in the bulk of the embryo. We propose that piRNAs and their associated proteins act together with Smg to recruit the CCR4 deadenylation complex to specific mRNAs, thus promoting their decay. Because piRNAs involved in this regulation are produced from transposable elements, this identifies a direct developmental function for transposable elements in the regulation of gene expression.

In Drosophila embryos, Nos is expressed as a gradient which emanates from the posterior pole and organizes abdominal segmentation¹⁵. The majority of nos mRNA is distributed throughout the bulk cytoplasm, translationally repressed¹⁶, and subsequently degraded during the first 2–3 hours of development. This repression is essential for head and thorax segmentation^16,17. A small amount of nos transcripts, localised at the posterior pole of the embryo, escapes degradation and is actively translated, giving rise to the Nos protein gradient. nos mRNA decay in the bulk cytoplasm depends on the CCR4-NOT deadenylation complex and its recruitment onto nos by Smg. This contributes to translational repression in the bulk of the embryo and is required for embryonic antero-posterior patterning¹³.

Smg was suggested not to be the only activator of nos mRNA decay during early embryogenesis^11,12. Zygotically-expressed miRNAs have been reported to activate maternal mRNA deadenylation in zebrafish embryos¹⁸ and decay in Drosophila embryos¹⁴. We investigated the potential involvement of other classes of small RNAs in mRNA deadenylation and decay before zygotic expression. Since piRNAs are expressed maternally in the germline and present in early embryos^19,20, we analysed the possible role of the piRNA pathway in maternal mRNA deadenylation. Piwi, Aub and Ago3 are specific Argonaute proteins^1,3,21,22, Armitage (Armi) and Spindle-E (Spn-E) are RNA helicases, and Squash (Squ) is a nuclease^2,10,23,24 involved in piRNA biogenesis and function. Poly(A) test (PAT) assays were performed to measure nos mRNA poly(A) tail length in embryos spanning one hour intervals during the first four hours of embryogenesis. In contrast to the progressive shortening of nos mRNA poly(A) tails observed in wild-type embryos correlating with mRNA decay during this period, nos poly(A) tail shortening was affected in embryos from females mutant for the piRNA pathway (herein referred to as mutant embryos) (Figure 1a, Supplementary Figures 1a, 2, 12). This defect in deadenylation correlated with higher amounts of nos mRNA in mutant embryos, as quantified by RT-QPCR (Figure 1b). in situ hybridization revealed stabilized nos mRNA in the bulk cytoplasm of mutant embryos where it is normally degraded in the wild-type (Figure 1c, Supplementary Figure 1b). Consistent with previous data showing that nos mRNA deadenylation is required for translational repression¹³, defective deadenylation in mutant embryos resulted in the presence of ectopic Nos protein throughout the embryo (Figure 1d, Supplementary Figure 1c). The presence of Nos in the anterior region results in the repression of bicoid and hunchback mRNA translation and in affected head skeleton. Consistent with earlier mentioned defects⁷, we found that the piwi¹ mutant embryos which were able to produce a cuticle had strong head defects (Figure 1e).

The piRNA pathway plays a role during early oogenesis in preventing DNA damage, possibly through the repression of transposable element transposition. DNA double-strand breaks arising in mutants of the piRNA pathway result in affected embryonic axis specification, and this developmental defect is suppressed by mutations in the Chk2 DNA damage signal transduction pathway^9,10. We found that defects in nos mRNA deadenylation and decay observed in aub or armi mutants were not suppressed by Chk2 (mnk^P6) mutations, indicating that these defects did not result from activation of the Chk2 pathway earlier during oogenesis (Supplementary Figure 3a–c). Moreover, affected deadenylation of nos mRNA in piRNA pathway mutants did not depend on oskar (Supplementary Figure 3d).

We addressed a potential direct role of the piRNA pathway in the regulation of nos mRNA deadenylation and decay in the embryo. Aub and Piwi accumulate in the pole plasm and in pole cells of the embryo^25,26. However, we found lower levels of Aub and Piwi throughout the entire embryo (Figure 2a, Supplementary Figures 4, 5). Ago3 was also present throughout the embryo (Supplementary Figure 6a, c). Aub and Ago3 were cytoplasmic and accumulated in discrete foci, a distribution similar to those of CCR4 and Smg (Figure 2b, Supplementary Figure 6b). CCR4 and Smg were reported to partially colocalise in small cytoplasmic foci¹³. Aub and Ago3 also partially colocalised with Smg and CCR4 in the bulk of syncytial embryos, in both cytoplasmic foci and a diffusely distributed cytoplasmic pool (Figure 2b, Supplementary Figure 6b). Importantly, the distributions of CCR4 and Smg depended on the piRNA pathway, as they were strongly affected in aub and spn-E mutant embryos. Although global amouts of CCR4 and Smg did not decrease in mutant embryos, CCR4 foci strongly increased in size, whereas Smg foci decreased in size or disappeared (Figure 2c, d). This suggests different functions for a subset of CCR4 and Smg foci and that deadenylation could take place diffusely in the cytoplasm. These results demonstrate a functional link between CCR4-mediated deadenylation and the piRNA pathway.

Co-immunoprecipitation experiments showed that Aub co-precipitated Smg, CCR4 and Ago3 in the absence of RNA, suggesting the presence of these proteins in a common complex (Figure 3a, Supplementary Figure 7a, b). Smg also co-precipitated CCR4, Aub and Ago3 (Figure 3b, Supplementary Figure 7c). Piwi, however, was not found to co-precipitate Smg or CCR4 (data not shown). Importantly, Smg, CCR4 and Ago3 also co-precipitated with Aub in osk⁵⁴ mutant embryos that are defective in pole plasm assembly²⁷, indicating the presence of this complex outside the pole plasm (Figure 3a). We next showed that nos mRNA co-precipitated with Aub in both wild-type and osk⁵⁴ embryos. The amounts of nos mRNA were similar in Aub and Smg immunoprecipitates (Figure 3c).

These findings show that the Argonaute proteins Aub and Ago3 associate with Smg and the CCR4 deadenylase complex to directly regulate nos mRNA in the bulk cytoplasm of early embryos.

nos 3′-UTR contains Smg binding sites (SRE) located in its 5′-most region (referred to as TCE)¹⁶. We searched for piRNAs, sequenced from early embryos, presumed capable of targeting nos 3′-UTR based on their sequence complementarity. Strikingly, a specific region located in the 3′-most part of the 3′-UTR could be targeted by over 200 copies of piRNAs originating from two transposable elements, 412 and roo (Figure 4a, Supplementary Figure 8). piRNAs complementary to nos 3′-UTR were visualized by northern blots. In addition, piRNAs predicted to target nos 3′-UTR co-immunoprecipitated with Aub (Figure 4b). We used nos genomic transgenes deleted for different parts of the 3′-UTR¹⁶ to address the requirement of the corresponding regions for nos mRNA deadenylation. We have shown previously that the TCE (nt 1–184) is required for nos mRNA poly(A) tail shortening, consistent with the role of Smg in this process¹³. Deletion of region 184–403 (nos(Δ1)) had no effect, whereas poly(A) tails from the transgene deleted of the region 403–618 (nos(Δ2)) were elongated in 3–4 hour embryos (Figure 4c, Supplementary Figure 12). This could indicate a regulation by the miRNA predicted by miRBase to target this region. Deletion of 618–844 in nos 3′-UTR (nos(Δ3)) had a strong effect on nos deadenylation (Figure 4c, Supplementary Figures 9, 12). Consistent with this, nos mRNA levels produced by this transgene remained mostly stable (Figure 4d). This resulted in defects in embryo patterning: A total of 35% (n=1894) of embryos from nos(Δ3) females did not hatch and among them 86% (n=28) showed head skeleton defects (Figure 3e). We next deleted specific sequences complementary to 412 (15 nt) and roo (11 nt) retrotransposon piRNAs (Supplementary Figure 8). These short deletions, either independently or in combination, affected nos mRNA deadenylation (Figure 4f, Supplementary Figure 12).

To further support the role of retrotransposon piRNAs in nos mRNA regulation, we blocked 412 and roo piRNAs by injecting specific 2′ O-methyl anti-piRNA in embryos²⁸, and recorded cuticles as a functional assay of Nos ectopic synthesis at the anterior pole. Injection of anti-piRNA(412) or anti-piRNA(roo) resulted in specific head development defects (Figure 4g).

Together, these results provide strong evidence that an interaction between piRNAs and nos mRNA is required for nos mRNA deadenylation and translational repression in the first hours of embryogenesis.

We have identified a new function of the piRNA pathway in the regulation of maternal mRNAs. Recently, piRNAs derived from 3′-UTR of cellular transcripts have been identified in gonadal somatic cells, although their biological role has not been clarified^29,30. Here, we propose that piRNAs, in complex with Piwi-type Argonaute proteins Aub and Ago3, target nos maternal mRNAs and recruit or stabilize the CCR4-NOT deadenylation complex together with Smg (Supplementary Figure 10). These interactions induce rapid mRNA deadenylation and decay. Thus, activation of mRNA deadenylation represents a new direct mechanism of action for the piRNA pathway with an essential developmental function during the first steps of embryogenesis.

Smg is a general factor for mRNA decay during early embryogenesis¹². Because Aub and Ago3 are present in a complex with Smg in early embryos, a proportion of Smg mRNA targets could be regulated by the piRNA pathway. Consistent with this, other maternal mRNAs that are destabilized during early embryogenesis, are targeted by abundant piRNAs and their deadenylation depends on the piRNA pathway (Supplementary Figure 11).

These piRNAs involved in gene regulation are generated from transposable element sequences. Although transposable elements have been described to be essential for genome dynamics and evolution, their immediate function within an organism has remained rather elusive. This study provides evidence for a co-evolution between transposable elements and the host genome and reveals the direct developmental function of transposable elements in embryonic patterning, through the regulation of gene expression.