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

Abstract

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 and transcriptional and post-transcriptional silencing. The piRNA pathway has other essential functions in germline stem cell maintenance and in maintaining germline DNA integrity. Here we uncover an unexpected function of the piRNA pathway in the decay of maternal messenger RNAs 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 and the deadenylase CCR4, as well as the zygotic expression of a microRNA cluster. 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 the nos 3' untranslated region. 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, one of the Argonaute proteins in the piRNA pathway, is present in a complex with Smaug, CCR4, nos mRNA and piRNAs that target the nos 3' untranslated region, in the bulk of the embryo. We propose that piRNAs and their associated proteins act together with Smaug to recruit the CCR4 deadenylation complex to specific mRNAs, thus promoting their decay. Because the 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.

Figure 1

The piRNA pathway is required for nos mRNA deadenylation and decay as well as translational repression in the bulk cytoplasm of the embryo. (a, b) PAT assays and RT-QPCR of nos mRNA. Mutant females of the indicated genotypes were crossed with wild-type males. The sop mRNA was used as a control in a. (b) Levels of nos mRNA in 2–3 hour and 3–4 hour embryos. Mean value of three quantifications, error bars correspond to s.d. (c) In situ hybridizations of nos mRNA. (d) Immunostaining of embryos with anti-Nos antibody. (e) Cuticle preparations of piwi¹ embryos showing head defects (rudimentary head skeleton (top panel), head skeleton replaced by a hole (bottom panel)). 2% of embryos from piwi¹ germline clones produced a cuticle (n=1060), among those, 22/23 had strong head defects. No embryos from aub^N11/aub^HN2 (n=1230) or aub^QC42/aub^HN2 (n=813) females produced a cuticle.

Figure 2

Aub is present in the bulk of the embryo and the piRNA pathway is required for CCR4 and Smg cytoplasmic distributions. (a) Confocal images of cytoplasmic expression of Aub in the embryo. Syncytial blastoderm embryo at nuclear cycle 11, anterior is to the left. Pole cells of the same embryo, at the same setting (middle Aub panel) and at lower intensity (right Aub panel)^,. DAPI staining (right panel). (b) Double immunostaining of embryos at nuclear cycles 11/12 with anti-Aub and anti-Smg, or anti-Aub and anti-CCR4. Arrows indicate examples of small foci showing colocalisation in b and c. (c) Smg and CCR4 cytoplasmic distributions are affected in aub and spn-E mutant embryos. Double immunostaining of embryos at nuclear cycle 11 with anti-CCR4 and anti-Smg. (d) Western blots of proteins from 0–2 hour embryos revealed with anti-Smg and anti-CCR4. α-tubulin (Tub) was used as a loading control.

Figure 3

Aub, Ago3, Smg, CCR4 and nos mRNA are present in a common complex in the bulk of the embryo. (a) Co-immunoprecipitations of Smg, CCR4 and Ago3 with Aub in 0–2 hour embryo extracts. Anti-Aub and anti-GFP were used for immunoprecipitations in wild-type, osk⁵⁴ and GFP-Aub expressing embryos, respectively. (b) Co-immunoprecipitations of CCR4, Aub and Ago3 with Smg in 0–2 hour wild-type embryo extracts. The asterisks indicate immunoglobulins. (c) Quantification of nos mRNA enrichment in Aub and Smg immunoprecipitations. Extracts from 0–2 hour wild-type or osk⁵⁴ embryos were immunoprecipitated with anti-Aub (rabbit), or anti-Smg. For quantifications performed by RT-QPCR, the ratio of nos mRNA/rp49 mRNA was set to 1 in the mock immunoprecipitation. Mean value of three quantifications, error bars correspond to s.d. rp49 was used as a control mRNA.

Figure 4

piRNAs target a specific region in nos 3′-UTR which is required for nos mRNA deadenylation. (a) Schematic representation of nos 3′-UTR. The regions deleted in nos genomic transgenes (nos(Δ)) are indicated on the upper line. SRE, piRNA and miRNA target sites are indicated. Predictions of miRNA targeted regions are from miRBase (miR-31a, miR-314 and miR-263b from proximal to distal). piRNA occurrences in the data sets^, are indicated. (b) Northern blots of 0–2 hour embryos probed with riboprobes corresponding to sense nos 3′-UTR (position 403–844) (left panel) and to antisense 412 piRNA (right panel). Anti-GFP immunoprecipitations (GFP IP) were performed using wild-type and GFP-Aub expressing embryos. (c) nos PAT assays. For nos(Δ3) the fragment amplified in the PAT assay is shorter than the fragment amplified in the other nos PAT assays (Supplementary Figure 9). (d) Quantification of nos mRNA levels from the nos(Δ3) transgene by RT-QPCR. Mean value of three quantifications, error bars correspond to s.d. (e) Cuticle preparations of embryos from nos(Δ3) females (lack of head skeleton). (f) PAT assays of embryos with nos genomic transgenes in which sequences complementary to 412 piRNA, roo piRNA, or both sequences have been deleted. The sop mRNA was used as a control in c and f. (g) Injection of 2′ O-methyl anti-piRNA in embryos. Control injections were with injection buffer alone or with the irrelevant anti-miR129. Examples of cuticles following injections of anti-miR129 (wild-type head skeleton), anti-pi(412) and anti-pi(roo) (affected head skeleton).