Germline inherited small RNAs clear untranslated maternal mRNAs in C. elegans embryos

Inheritance and clearance of maternal mRNAs are two of the most critical events required for animal early embryonic development. However, the mechanisms regulating this process are still largely unknown. Here, we show that together with maternal mRNAs, C. elegans embryos inherit a complementary pool of small non-coding RNAs capable of triggering the cleavage and removal of hundreds of maternal mRNAs. These antisense small RNAs are loaded into the maternal catalytically-active Argonaute CSR-1 and cleave complementary mRNAs no longer engaged in translation in somatic blastomeres. Induced depletion of CSR-1 specifically during embryonic development leads to embryonic lethality in a slicer-dependent manner and impairs the degradation of CSR-1 embryonic mRNA targets. Given the conservation of Argonaute catalytic activity, we propose that a similar mechanism operates to clear maternal mRNAs during the maternal-to-zygotic transition across species.


MAIN TEXT
The elimination of germline-produced mRNAs and proteins in somatic blastomeres and the concomitant activation of the zygotic genome-the maternal to zygotic transition (MZT)-is critical for embryogenesis 1 . Despite several mechanisms have been discovered to regulate the clearance of maternal mRNAs during the MZT, they only account for the regulation of a small fraction of decayed transcripts, suggesting that factors and pathways responsible for the decay of the majority of maternal mRNAs still remain elusive. In addition, distinct species can adopt different strategies to achieve the clearance of maternal mRNAs. This is why understanding how different species clear maternal mRNAs is fundamental to shed light on novel mechanisms regulating of one the most important and conserved transition happening at the beginning of embryogenesis. For instance, small RNAs, such as maternally-inherited PIWI-interacting RNAs (piRNAs) in Drosophila 2,3 and zygotically-transcribed micro RNAs (miRNAs) in Drosophila, zebrafish and Xenopus [4][5][6] have been shown to directly regulate the degradation of maternal mRNAs in embryos. However, miRNA functions are globally suppressed in mouse oocytes and zygotes 7,8 and they do not appear to contribute to maternal mRNA clearance in C. elegans embryos 9 . This raises the question of whether other type of small RNAs might play a role in this process. In this regard, catalytically active Argonaute and endogenous small interfering RNAs (endo-siRNAs) have been proposed to play an essential role in oocytes and early embryos in place of miRNAs 10,11 .
Therefore, in addition to the silencing of repetitive elements, the RNA interference (RNAi) pathway might regulate the removal of maternal mRNAs in animal embryos.
In the nematode C. elegans, two waves of maternal mRNA clearance have been documented so far. The first wave of maternal mRNA clearance, regulated by a consensus sequence in the 3'UTR, occurs during the transition from the oocyte to the 1-cell embryo 9 . A second wave of clearance occurs in the somatic blastomeres of the developing embryos 12,13 . However, to date no mechanisms are known to regulate this process in the embryo. Endo-siRNAs produced in the germline are known to be heritable and they can potentially regulate mRNA transcripts in the developing embryos. Two main endogenous small RNA pathways regulate heritable epigenetic processes in the C. elegans germline: (1) thousands of PIWI-interacting RNAs (piRNAs) that target foreign "non-self" mRNAs 14 , and (2) endogenous antisense small RNAs that target active "self" mRNAs 15,16 . These antisense small RNAs, called 22G-RNAs, are generated by the RNA-dependent RNA polymerase (RdRP) EGO-1 using target germline mRNAs as a template and are then loaded into the Argonaute CSR-1 (Chromosome-Segregation and RNAi deficient) 16,17 . CSR-1 22G-RNAs are thought to protect germline mRNAs from piRNA silencing and they are essential for fertility and embryonic development 15,18,19 . However, CSR-1 is also responsible for the majority of slicing activity in C. elegans extracts and is capable of cleaving complementary mRNAs in vitro 20 .
Moreover, CSR-1 slicer activity is capable of fine-tuning some of its mRNA targets in the adult germline 21 . Therefore, whether CSR-1 can protect or degrade its germline mRNA targets is still an open question.
Here, we provide novel insight into the widespread process of maternal mRNA clearance in early developing embryos. We demonstrate that 22G-RNAs loaded into CSR-1 that are inherited from the maternal germline, trigger the cleavage and removal of complementary maternal mRNAs in somatic blastomeres during early embryogenesis. Whereas in the germline CSR-1 and its associated 22G-RNAs result in a modest fine-tuning of target mRNAs (Gerson-Gurwitz et al., 2016), in the embryo CSR-1 targets are cleared. We found that target mRNAs that decay due to CSR-1 are depleted of ribosomes in embryonic somatic cells, and we provide evidence that the translational status of these maternal mRNAs influences their decay. We employed the auxininducible degradation (AID) system 22 to deplete CSR-1 specifically during the MZT to study its functions in early embryos. Using this approach, we demonstrate that the slicer activity of CSR-1 is essential for embryonic viability and is required to post-transcriptionally cleaves its embryonic mRNA targets. Given the conservation of Argonaute catalytic activity in metazoans and the conserved function of small RNAs in regulating maternal mRNA clearance in several animal models we propose that a similar mechanism operates to clear maternal mRNAs during the MZT across species.

CSR-1 localizes to the cytoplasm of somatic blastomeres and its slicer activity is essential for embryonic development.
To study CSR-1 localization during embryogenesis, we have generated various CRISPR-Cas9 tagged versions of CSR-1 23 . CSR-1 is expressed mainly in the germline of adult worms 17 and localizes in the cytoplasm and germ granules (Extended Data Fig. 1a, b). However, in early embryos, CSR-1 localizes both in the germline and somatic blastomeres (Fig. 1a, Extended Data Fig. 1b). CSR-1 in the somatic blastomeres is mainly cytoplasmic and persists for several cell divisions (Fig. 1a, Extended Data Fig. 1b). This is in contrast with other germline Argonaute proteins 24,25 , such as PIWI, which exclusively segregate with the germline blastomere from the first embryonic cleavage (Fig. 1a, Extended Data Fig. 1b). Animals lacking CSR-1 or expressing a CSR-1 catalytic inactive protein show several germline defects, severely reduced brood sizes, chromosome segregation defects, and embryonic lethality 16,21,26 .
To understand the role of CSR-1 during embryogenesis, we depleted CSR-1 protein using the auxin-inducible degradation (AID) system 22 (Extended Data Fig. 3a, b). First, we depleted CSR-1 from the first larval stage (L1), recapitulating the fertility loss and embryonic lethality phenotypes of both csr-1 (tm892) mutant and catalytically-inactive CSR-1 mutant worms 21,26 (Fig.   1b, c, and Extended Data Fig. 2a, b). Next, we depleted CSR-1 from the beginning of oogenesis 6 (Extended Data Fig. 3a, b). This treatment did not significantly decrease the fertility of adult worms ( Fig. 1b), yet all the CSR-1-depleted embryos failed to develop in larvae (Fig. 1c), suggesting CSR-1 plays an essential role during embryogenesis.
To test whether the catalytically active CSR-1 is required for embryonic viability during the MZT, we expressed a single transgenic copy of CSR-1, using MosSCI 27 , with or without an alanine substitution of the first aspartate residues within the catalytic DDH motif and depleted endogenous CSR-1 at the beginning of oogenesis (Fig. 1d). The CSR-1 catalytic dead mutant (ADH) failed to suppress the embryonic lethality caused by the depletion of endogenous CSR-1 (Fig. 1d), whereas catalytically active CSR-1 (DDH) significantly restored embryonic viability (Fig. 1d). Thus, CSR-1 slicer activity is essential during embryogenesis.

CSR-1 post-transcriptionally regulates its targets in the embryo.
The localization of maternally inherited CSR-1 in the somatic blastomeres during early embryogenesis suggests that CSR-1 and its interacting 22G-RNAs may regulate specific embryonic targets in somatic cells. To better understand the regulatory function of CSR-1 in the embryo, we identified and compared CSR-1 -interacting 22G-RNAs in the embryo and adult worms. CSR-1 was immunoprecipitated in embryo populations ranging from 1-to 20-cells stage (Extended Data Fig. 4a) or young adult worms, followed by sequencing of interacting 22G-RNAs.
CSR-1-22G-RNA targets in the adult germlines and early embryos largely overlap (74 %) (Fig.   2a). However, the relative abundance of the 22G-RNAs complementary to these targets was different between the adult germlines and early embryos (Fig. 2a, b). Only 29 % of CSR-1 targets with the highest levels of complementary 22G-RNAs (> 150 reads per million, RPM) in adults and embryos are shared (Fig. 2a, b). Given that CSR-1 cleavage activity on target mRNAs depends on the abundance of 22G-RNAs 21 , our results suggest that CSR-1 may regulate a different subset of mRNAs in the embryos.
To evaluate gene expression changes upon CSR-1 depletion in embryos, we depleted CSR-1 from the beginning of oogenesis and collected early embryo populations fully depleted of CSR-1 (Extended Data Fig. 3a, and Extended Data Fig. 4b, c). Analysis of nascent transcription using global run-on sequencing (GRO-seq) revealed that the zygotic transcription in CSR-1 depleted embryos was largely unaffected compared to control untreated embryos (Fig. 2c, Extended Data

CSR-1 cleaves maternal mRNAs in early embryos.
In the first phases of embryogenesis, the embryo is transcriptionally silent and relies on maternally inherited mRNA transcripts. Maternal mRNAs degradation is concomitant with the zygotic genome activation, during the MZT 1 . To gain insight into the type of mRNA targets regulated by CSR-1, we asked whether CSR-1 embryonic targets corresponded to maternal or zygotic mRNA transcripts. We adapted a previously developed sorting strategy 28 to sort embryo populations at the 1-cell stage (99 % pure), or enrich for early embryos (from 4-to 20-cells), or late embryos (more than 20-cells) (Extended Data Fig. 4d). Strand-specific RNA-seq was performed on these embryo populations to profile gene expression dynamics. We detected mRNAs from 7033 genes in 1-cell embryos (transcript per million, TPM, > 1) (Supplemental Table 1), which we classified into three groups based on their dynamics during embryonic development.
"Maternal cleared" mRNAs (1320 genes) corresponded to maternal mRNAs in 1-cell embryos whose levels diminished in early and late stages (Fig. 3a) (Supplemental Table 1). These genes belong to the previously characterized Maternal class II genes, which are maternally inherited in the embryos and degraded in somatic blastomeres 12 . "Maternal stable" mRNAs (1020 genes) corresponded to maternal mRNAs in 1-cell embryos whose levels are stably maintained in early and late embryos (Fig. 3a) (Supplemental Table 1). "Zygotic" mRNAs (704 genes) whose mRNAs accumulated in early and late embryos but were undetectable in 1-cell embryos (TPM < 1) ( Fig.   3a) (Supplemental Table 1).
Analysis of the embryonic CSR-1 targets revealed that they were inherited in 1-cell embryos and gradually depleted in early and late embryos, similarly to the maternal cleared class of genes ( Fig. 3a), suggesting that CSR-1 contributes to maternal mRNA clearance in somatic blastomeres.
To validate this result, we analyzed the levels of maternal cleared mRNAs in CSR-1 depleted embryos and found accumulation of maternal cleared mRNA targets correlated with the abundance of antisense 22G-RNAs loaded onto CSR-1 (Fig. 3b). To determine if CSR-1 slicer activity is required to regulate those targets we measured gene expression changes in CSR-1 depleted embryos complemented with transgenic expression of CSR-1 catalytic dead mutant (ADH) or catalytically active CSR-1 (DDH), and found increased accumulation of CSR-1 targets in slicerinactive CSR-1 embryo population (Fig. 3c). Finally, we performed RNA-seq on CSR-1 depleted in 1-to 4-cells stage enriched embryos or 4-to 20 cell stage enriched embryos (Extended Data Fig.   5a-c) to study if CSR-1 is directly regulating these maternal mRNA targets in embryos. CSR-1 depletion in 1-to 4-cell embryos did not cause global changes in maternal cleared mRNA target levels (Extended Data Fig. 5d). However, CSR-1 depletion in 4-to 20-cell stage enriched embryos resulted in increased levels of maternal cleared mRNA targets compared to 1-to 4-cell embryos (Extended Data Fig. 5d). Thus, CSR-1 and its interacting 22G-RNAs actively degrade their maternal mRNA targets during early embryogenesis. To verify these results, we performed RNA single-molecule fluorescence in situ hybridization (smFISH) on a maternal cleared CSR-1 mRNA target in CSR-1 depleted and control embryos. We observed reduced maternal mRNA degradation of the CSR-1 target in somatic blastomeres of 20-cell stage embryos depleted of CSR-1 compared to control (Fig. 3d, Extended Data Fig. 5e). Collectively, these results suggest that the maternally inherited CSR-1 loaded with 22G-RNAs contribute to cleave and degrade complementary maternal mRNA targets in somatic blastomeres.

CSR-1 preferentially targets maternal mRNAs no longer engaged in translation.
To analyze the dynamics of CSR-1-dependent mRNA clearance during embryogenesis, we divided maternally inherited mRNAs into those degraded early and late during embryogenesis. that the levels of 22G-RNAs targeting the early-degraded mRNAs were significantly higher compared to late-degraded mRNAs in total RNAs and CSR-1 IPs (Extended Data Fig. 6a, b). These results suggest that the abundance of 22G-RNAs correlates with the temporal decay of maternal cleared mRNAs during embryogenesis.
We generated metaprofiles for the levels of CSR-1-interacting 22G-RNAs levels along the gene body of early-and late-degraded mRNA targets (Fig. 4c, d). The early-degraded mRNA genes showed an enrichment of CSR-1-loaded 22G-RNAs along the whole coding sequence (Fig. 4b, c).
The late-degraded mRNA genes showed enrichment of CSR-1-loaded 22G-RNAs mainly at the 3' end, corresponding to the 3'UTR ( Fig. 4b, d). Previous reports have shown that the rate of mRNA clearance during maternal to zygotic transition is influenced by translational efficiency 29,30 . Thus, the different translational status of early-and late-degraded mRNAs may influence the accessibility and production of CSR-1-loaded 22G-RNAs antisense to the coding sequences of cleared mRNAs.
To test this hypothesis, we measured ribosomal occupancy by Ribo-seq in a population of early embryos (Extended Data Fig. 6c). Early-degraded mRNAs showed significantly lower ribosomal occupancy and translational efficiency compared to late-degraded mRNAs (Fig. 4e, f). These results suggest that ribosomal loading on maternal mRNAs can influence their coding sequence accessibility for the 22G-RNA-mediated cleavage by CSR-1 in somatic blastomeres.

Ribosome occupancy affects CSR-1-mediated maternal mRNA clearance.
Previous studies have shown that mRNA codon usage can influence translational efficiency and maternal mRNA decay during the MZT 29,30 . However, the early-and late-degraded mRNAs did not show any differences in their codon usage (Extended Data Fig. 6d, e). Because the translation of germline mRNAs is primarily regulated by their 3'UTR in C. elegans 31 , we investigated whether different 3'UTRs from early-and late-degraded mRNAs can impact translation and mRNA degradation in early embryos. We generated two single-copy transgenic lines expressing a germline mCherry::h2b mRNA reporter with either a 3'UTR from an early-(egg-6 3'UTR) or a late-(tbb-2 3'UTR) degraded mRNA target (Fig. 5a). The steady-state level of the two mCherry::h2b mRNA reporters was similar in adult worms (Fig. 5b), whereas the level of mCherry::h2b mRNA fused with the early-degraded egg-6 3'UTR was lower in embryo, indicating increased degradation in early embryos (Fig. 5b). To verify that the different 3'UTRs affect the translational efficiency of mCherry::h2b mRNA in embryos we quantified the ribosome occupancy by Ribo-seq and mRNA level by RNA-seq, and observed decreased translational efficiency of mCherry transgenic reporter with egg-6 3'UTR ( Fig. 5c, Extended Data Figs. 6f and 7a, b). To test whether CSR-1 22G-RNAs contribute to the increased decay of the mCherry::h2b reporter fused to the egg-6 3'UTR, we sequenced small RNAs in early embryo preparations from the two transgenic lines (Extended Data Fig. 6f). We observed increased levels of small RNAs on the coding sequences of the mCherry transgenic reporter with egg-6 3'UTR compared to the tbb-2 3'UTR reporter (Fig. 5d, e). Thus, the translation level of maternal mRNAs can influence the rate of CSR-1-mediated mRNA cleavage in somatic blastomeres.
In summary, our study identifies a novel function of the conserved Argonaute slicer activity in the degradation of maternally inherited mRNAs during the MZT.

DISCUSSION
In this study, we have shown that the maternally inherited CSR-1 protein and its interacting 22G-RNAs trigger the cleavage of hundreds of complementary maternal mRNA targets in embryos. Despite most of the inherited Argonautes localize to the germline blastomeres, the inherited CSR-1 also localizes to the cytoplasm of somatic blastomeres for several cell divisions during early embryogenesis. We have shown that C. elegans embryos inherit a pool of 22G RNAs, loaded onto CSR-1, antisense to mRNAs that undergoes rapid degradation in developing embryos, and CSR-1 slicer activity facilitates the clearance of these maternal mRNAs in somatic blastomeres. We have observed that CSR-1 is preferentially loaded with 22G-RNAs antisense to the coding sequences of untranslated mRNA targets in embryos. Based on our results, we propose that CSR-1 slicer activity is required during embryogenesis to cleave untranslated maternal mRNAs accumulated in somatic blastomeres after the first embryonic divisions. We speculate that active translation prevents the accessibility of CSR-1 22G-RNAs on complementary mRNA targets and hence the slicing through the coding sequences of translated maternal mRNAs (Fig. 6).
Therefore, the translation of maternal mRNAs ensures that CSR-1 22G-RNA complexes can only remove these mRNAs in the somatic cells of the developing embryos, when they are not needed to be translated anymore. This antagonism between translation and CSR-1 cleavage might explain why CSR-1 slicer activity results in only fine-tuning some of its complementary targets in the adult germlines. Thus, the presence of ribosomes on germline mRNAs may prevent the slicer activity of CSR-1, which is instead permitted in the embryos after the mRNAs are disengaged from translation and can be degraded. We have also shown that by modulating the translational activity of a maternal mRNA sequence we could detect different amounts of antisense 22G-RNAs. Therefore, it is possible that the presence of ribosome on the coding sequence of maternal mRNAs can antagonize the synthesis of 22G-RNAs by the RdRP.
It is known that translation affects maternal mRNA clearance, and recent works have shown how the codon usage of maternal mRNAs regulates translation and degradation rates in zebrafish embryos 29,30 . Here we show that in C. elegans embryos early and late degraded maternal mRNAs do not differ in their codon usage. Instead, we have observed that the 3'UTR is sufficient to regulate the translational efficiency of the coding sequence of a maternal mRNA in embryos, and in turn its degradation rate, including CSR-1 slicer activity. A consensus sequence in the 3'UTR of some maternal mRNAs has been shown to play a role in the degradation of mRNAs from oocytes to 1cell embryos in C. elegans 9 . Therefore, it would be interesting in the future to identify other consensus sequences located in the 3'UTR of maternal mRNAs and also RNA binding proteins that might contribute together with CSR-1 to regulate mRNA clearance during embryogenesis.
CSR-1 has been extensively studied in the adult germlines, where it has been proposed to protect active genes from piRNA silencing 18,19 , promote germline transcription 32,33 , regulate the biogenesis of histone mRNAs 34 , and fine-tune germline mRNAs loaded into oocytes 21 . The catalytic activity of CSR-1 and its gene regulatory function on target mRNAs has been proven be essential for fertility and chromosome segregations 21 . Similarly, our work provided evidences that CSR-1 slicer activity is essential for embryonic viability independently from its germline functions.
Therefore, we propose that the accumulation of maternal mRNAs in embryos lacking CSR-1 slicer activity might be deleterious for the developing embryo. Moreover, our finding suggests that mRNA translation in the germline might inhibit CSR-1 slicer activity on target mRNAs and results only in a mild fine-tuning of some targets. Therefore, we propose that the role of CSR-1 in protecting germline mRNA from piRNA silencing might be still compatible with its cleavage activity.
The requirement of Argonaute slicer activity to degrade endogenous target mRNAs has only been described in the silencing of repetitive elements in animals. However, catalytically active Argonaute proteins are conserved in animals, including humans. We propose that the RNAi activity of CSR-1 on maternal mRNAs might be a conserved mechanism required for the MZT and mRNA clearance across species. Even if miRNAs contribute to regulate maternal mRNA clearance in some animal models, including Drosophila, zebrafish and Xenopus, their functions are suppressed in mouse oocytes 7,8 . Instead, endogenous small RNAs targeting protein-coding genes, similar to the one loaded onto CSR-1, have been detected in mouse oocytes and embryonic stem cells [35][36][37] . This suggests that mammalian RNAi, in addition to roles in the suppression of repetitive elements, might also regulate endogenous genes. Given that the inheritance of maternal Ago2, the only catalytically active Argonaute in mouse, is essential for oocyte 10

COMPETING INTERESTS
All the authors declare no competing interests.

DATA AND MATERIALS AVAILABILITY
All the sequencing data are available at the following accession numbers GSE146062 (secure token for reviewers: krkpoycihjulhax). All other data supporting the findings of this study are available from the corresponding author on reasonable request.      26 Extended Data Fig. 2: csr-1(tm892) Fig. 2a, b. b, c, Degron::mCherry::3xFLAG::HA::CSR-1 embryos treated with auxin or ethanol (No Auxin) used for GRO-seq b, and RNA-seq c, in Fig. 2c, d, and Fig. 3b. d (a) or late-degraded (tbb-2 3'UTR) (b) mRNAs is shown above.

SUPPLEMENTAL TABLES
Supplemental Table 1: Gene lists generated and used in this study. Table 2: Strain lists generated and/or used in this study. Table 3: CRISPR-Cas9 guide RNA lists used in this study. Table 4: Primer lists used for RT-qPCR. Table 5: Oligos used for smFISH.

C. elegans strains and maintenance
Strains were grown at 20 °C using standard methods 38 . The wild-type reference strain used was Bristol N2. A complete list of strains used in this study is provided in Table S2.

3'UTR replacement experiment
A sequence of 800bp downstream of the stop codon of egg-6 was amplified from genomic DNA by PCR and used as 3' UTR. We used the single-copy transgene mCherry::his-11::tbb-2 3'UTR as entry strain to insert egg-6 3'UTR by CRISPR-Cas9 as described above. egg-6 3'UTR was inserted right after the STOP codon of his-11. The specific expression of the inserted UTR was validated by RT-qPCR. antibodies at a dilution of 1:500 and 1:800 respectively, and the secondary antibodies used were goat anti-mouse (Invitrogen, Alexa Fluor 488) and goat anti-rabbit (Invitrogen, Alexa Fluor 568) antibodies at a dilution of 1:500.

Auxin-inducible depletion of CSR-1
Auxin-inducible depletion has been performed as described in 22 . 250 mM Auxin stock solution was prepared in Ethanol and stored at 4°C. Auxin plates or Ethanol plates were prepared by the addition of Auxin or only Ethanol to NGM plates (final concentration: 500 µM auxin, 0.5 % Ethanol for Auxin plates and 0.5 % Ethanol for Ethanol plates). Plates were seeded with OP50 E.
Coli, stored at 4°C and warmed at room temperature before the experiment. Worms were placed on Auxin or Ethanol plates from L1 or at the beginning of oogenesis as explained for each experiment.

WT and CSR-1 mutants
Single L1 larvae were manually picked and placed onto NGM plates seeded with OP50 E. coli and grown at 20°C until adulthood and then transferred on a new plate every 24 hours for a total of 2 transfers. The brood size of each worm was calculated by counting the number of embryos and larvae laid on the 3 plates. For the auxin-depleted experiments NGM plates were supplemented with Ethanol (control) or Auxin.

Brood size of CSR-1 auxin-depleted worms during oogenesis
For CSR-1 depletion during oogenesis: brood size assay has been performed as described above with the exception that worms were grown from L1 on regular NGM plates and transferred onto Auxin or Ethanol plates 44h after hatching and transferred on new corresponding Auxin and Ethanol plates every 24 hours for a total of 2 transfers.

Embryonic lethality assay of CSR-1 rescue experiments
Single L1 larvae of strains carrying Degron::CSR-1 complemented with the single-copy insertion Mex-5P::GFP::CSR-1::tbb-2 3'UTR or Mex-5P::GFP::CSR-1(ADH)::tbb-2 3'UTR were manually picked and placed onto NGM plates seeded with OP50 E. coli and grown at 20°C until adulthood. Since transgene was prone to silencing, adult worms were allowed to lay at least 65 embryos and then adults were removed from the plates and imaged to check for GFP::CSR-1 expression. Only plates from GFP expressing adults were used for the assay. The percentage of embryonic lethality is calculated by dividing the number of dead embryos for the total number of laid embryos.

Collection of early embryos populations
Synchronous populations of worms were grown on NGM plates until adulthood and were carefully monitored using a stereomicroscope and bleached shortly after worms started to produce the first embryos. After bleaching, Early embryos were washed with cold M9 buffer to slow down embryonic development and immediately frozen in dry ice. A small aliquot of embryo pellet (2 µL) was taken right before freezing and mixed with 10 µL VECTASHIELD® Antifade Mounting Medium with DAPI (Vector laboratories) and immediately frozen in dry ice. DAPI stained embryos were defrosted on ice and used for counting cell nuclei and score the embryonic cell stage of the population.

Collection of CSR-1 depleted early embryos populations
Early embryos were collected as described above with the exception that worms were transferred on Auxin or Ethanol plates at the beginning of oogenesis.

Collection of early and late CSR-1 depleted embryo populations
Early populations of CSR-1 depleted embryos were collected as described above with the exception that harvesting was performed at the very beginning of embryo production to further enrich the population in early staged embryos. After bleaching, an aliquot of the same population was allowed to develop further for 1 to 2h in M9 containing 500 µM Auxin, 0.5 % Ethanol or 0.5 % Ethanol.
Embryonic cell stage was scored, and harvesting was performed when embryo populations reached the desired developmental stage.

Small RNA-seq library preparation
Total RNA from staged embryo preparations with RIN>9 was used to generate small RNA libraries. The library preparation was performed essentially as described previously 23 .

RNA IP
A synchronous population of 40,000 worms (48h after hatching) or a preparation of at least 150,000 early embryos was collected and suspended in extraction buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 5 mM MgCl2, 10 % glycerol, 0.25 % NP-40, protease inhibitor cocktails (Thermo Scientific), 40 U/mL RiboLock RNase inhibitors (Thermo Scientific)). Samples were crushed in a metal dounce on ice performing at least 40 strokes. Crude protein extracts were centrifuged at 12,000 rpm at 4°C for 10 minutes. Protein was quantified using Pierce™ 660 nm Protein Assay Reagent (Thermo Scientific) and 1 mg (for adults) or 700 µg (for embryos) of protein extract was used for RNA immunoprecipitation as described in 23 and used for sRNA-seq library preparation.

GRO-seq on CSR-1 depleted embryos
Populations containing at least 40,000 CSR-1 depleted early embryos were collected as described above. Early embryos were resuspended in 1.5 mL Nuclei extraction buffer (3 mM CaCl2, 2 mM MgCl2, 10 mM Tris HCl pH 7.5, 0.25 % Np-40, 10 % Glycerol, Protease inhibitors and RNase inhibitor 4U/mL) and transferred to a steel dounce and stroked 40 times. The lysate was cleared from cell debris by centrifuging at 100´g and nuclei were pellet at 1000´g and washed 4 times with Nuclei extraction buffer. Nuclei were washed once with Freezing buffer (50 mM Tris HCl pH 8, 5 mM MgCl2, 0.1 mM EDTA) and resuspended in 100 µL Freezing buffer.

Nuclear Run-On reaction and RNA extraction
Nuclear Run-On (NRO) reaction was performed by addition of 100 µL NRO 2x buffer (10 mM Tris HCl, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 1 % Sarkosyl, 0.5 mM ATP, CTP and GTP and 0.8 U/µL RNase inhibitor) and using 1 mM Bio-11-UTP final concentration and incubation for 5 minutes at 30°C. NRO reaction was stopped by the addition of TRIzol LS reagent (Ambion) and RNA extraction was performed following the manufacturer's instructions. Purified RNA was fragmented by the addition of reverse transcriptase buffer and incubation for 7 minutes at 95°C.

Biotin RNA enrichment
Biotinylated nascent RNAs were bound to 30 µL Dynabeads MyOne Streptavidin C1 (Invitrogen and) washed 3 times as described in 40 and purified with TRIZOL reagent.

Ligation of 3' and 5' Adapter Oligos and 2 nd and 3 rd Biotin enrichments
RNA was ligated to 3' end adapter using T4 RNA ligase 2 Truncated KQ (home-made) for 16h at 15˚C. After ligation RNA was purified using solid-phase reversible immobilization beads (SPRI beads) and biotinylated RNA was enriched as described above. After purification RNA was ligated at 5' end using T4 RNA ligase 1 for 2 h at 25˚C. RNA was purified using SPRI beads and biotinylated RNA was enriched for a third time as described above.

Reverse transcription and amplification of cDNA libraries
Purified RNA was reverse transcribed using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific) following manufacturer conditions except that reaction was incubated for 1h at 50˚C. cDNA was PCR amplified with specific primers using Phusion High fidelity PCR master mix 2x (New England Biolab) for 18-20 cycles and sequenced on Illumina Next 500 system.

Sorting of C. elegans embryos
Sorting of C. elegans embryos was performed as described in 28 with the following modifications: a strain expressing both mCherry::OMA-1 and PIE-1::GFP was used to collect embryos at three different developmental stages (see Extended Data Fig. 3c); embryos after bleaching were fixed with 2% formaldehyde in M9 to block cell division. After sorting, embryos were reverse crosslinked in 250 µL RIPA buffer with RNase inhibitors for 30 minutes at 70°C and RNA was extracted with TRIzol LS (Ambion) following manufacturer instructions.

Strand-specific RNA-seq library preparation
DNase-treated total RNA with RIN > 8 was used to prepare strand-specific RNA libraries. Strandspecific RNA-seq libraries were prepared as described previously 23 .

RT-qPCR
1 µg DNase treated total RNA was used as a template for cDNA synthesis using random hexamers and M-MLV reverse transcriptase. qPCR reaction was performed using Applied Biosystems Power up SYBR Green PCR Master mix following the manufacturer's instructions and using an Applied Biosystems QuantStudio 3 Real-Time PCR System. Primers used for qPCR are listed in Table S4.

Single-molecule FISH (smFISH)
Single-molecule FISH (smFISH) was performed as described in 41 . Briefly, embryos were harvested by bleaching, immediately resuspended in Methanol at -20˚C, freeze cracked in liquid nitrogen for 1 minute and incubated at -20˚C overnight. Embryos were washed once in wash buffer (10 % formamide, 2x SSC buffer (Ambion)) and hybridized with the corresponding FLAP containing probes in 100 µL hybridization buffer (10 % dextran sulfate, 2 mM vanadylribonucleoside complex, 0.02 % RNAse-free BSA, 50 µg E. coli tRNA, 2´ SSC, 10 % formamide) at 30˚C overnight. Hybridized embryos were washed twice with wash buffer and once in 2x SSC buffer before imaging. Right before imaging, embryos were resuspended in 100 µL anti-fade buffer (0.4 % glucose, 10 µM Tris-HCl pH8, 2x SSC) with 1 µL catalase (Sigma-Aldrich) and 1 µL glucose oxidase (3.7 mg/mL, Sigma-Aldrich) and stained with VECTASHIELD® Antifade Mounting Medium with DAPI (Vector laboratories). DAPI staining was used to select embryos at 20-cell stage. Images of the central plane of embryos at desired developmental stages were acquired on a Zeiss LSM 700 confocal microscopy. Oligos used for smFISH of C01G8.1 mRNA target are listed in Table S5.
The detection of single mRNA molecules was performed with the open-source Matlab package FISH-quant as previously described 42 . Briefly, RNA signal was enhanced by a 2-step convolution of the image with Gaussian Kernels. First, the image background obtained by convolution with a large Gaussian Kernel was estimated and then subtracted. Second, the resulting image was filtered with a small Gaussian Kernel to further enhance the signal-to-noise ratio. RNA spots were detected with a local maximum algorithm. For each embryo, a manually drawn outline was used to limit the detection to the somatic blastomeres and exclude the germline blastomere.

Ribo-seq
Ribo-seq has been performed as described in 43 with some modifications. Briefly, embryos harvested by bleaching were lysed by freeze grinding in liquid nitrogen in Polysome buffer (20 mM Tris-HCl pH 8, 140 mM KCl, 5 mM MgCl2, 1 % Triton X-100, 0.1 mg/mL cycloheximide) and 1 mg extract was digested by RNase I (100 U) at 37°C for 5 min and then fractionated on sucrose gradient (10-50 %) by ultracentrifugation at 39,000 rpm in a SW41-Ti rotor (Beckman coulter). RNA from monosome fraction was DNase treated and fragments of 28-30 nucleotides were size selected after running on a 15 % TBE-Urea gel. 28-30 nucleotide Ribosome protected fragments (RPF) were cloned with the previously described sRNA-seq library preparation approach with the following modifications: 3'phosphate was removed and 5' end was phosphorylated by treating RNA with Polynucleotide kinase.