Silencing of transposable elements (TEs) in the metazoan germline is critical for genome integrity and is primarily dependent on Piwi proteins and associated RNAs, which exert their function through both transcriptional and posttranscriptional mechanisms. Here, we report that the evolutionarily conserved Pelo (Dom34)‐Hbs1 mRNA surveillance complex is required for transposon silencing in the Drosophila germline. In pelo mutant gonads, mRNAs and proteins of some selective TEs are up‐regulated. Pelo is not required for piRNA biogenesis, and our studies suggest that Pelo may function at the translational level to silence TEs: This function requires interaction with Hbs1, and overexpression of RpS30a partially reverts TE‐silencing defects in pelo mutants. Interestingly, TE silencing and spermatogenesis defects in pelo mutants can also effectively be rescued by expressing the mammalian ortholog of Pelo. We propose that the Pelo‐Hbs1 surveillance complex provides another level of defense against the expression of TEs in the germline of Drosophila and possibly all metazoa.
The Pelo (Dom34)‐Hbs1 mRNA surveillance complex is required for transposon silencing in the Drosophila germline, which reveals a novel mechanism of transposon silencing possibly at the translational level.
Loss of Pelo causes up‐regulation of specific transposable elements (TEs) in both ovaries and testes.
Pelo is not required in the piRNA pathway.
Pelo functionally interacts with Hbs1 in TE silencing, and RpS30a overexpression partially rescues TE silencing defects in pelo mutants.
The Drosophila Piwi proteins and associated piRNAs defend against transposable elements (TEs) in the germline at both transcriptional and posttranscriptional levels . In the cytoplasm, the transcripts are subjected to endonucleolytic cleavage by RNA‐induced silencing complexes (RISCs) containing Aub or Ago3, two Piwi family proteins that generate piRNAs in the ping‐pong cycle to amplify the response to the actively transcribed TEs , . In the nucleus, the Piwi‐containing RISCs are guided to the chromatin via piRNAs to establish repressive chromatin state at the transposable DNA elements and other targeting loci , , . Murine members of Piwi and associated piRNAs are found to be associated with ribosomes and regulate translation during spermatogenesis , , but whether TEs could be regulated at the translational level is unknown.
Piwi, the founding member of the Drosophila piRNA pathway, was initially identified as an essential regulator of germline stem cell maintenance in the Drosophila ovary . Later studies demonstrate that Piwi is required in both the somatic niche cells and the germline for the maintenance, proliferation, and differentiation of germline stem cells , , . Pelo encodes a eukaryotic release factor (eRF1)‐like protein, and similar to piwi mutants, pelo homozygous null females have rudimentary ovaries because of its requirement for germline stem cell maintenance . Insights on the molecular function of Pelo came from yeast in which Dom34, the Pelo ortholog, is found to form a complex with a small GTPase Hbs1 to regulate endonucleolytic cleavage of mRNAs whose secondary structure causes ribosome stalling during translational elongation, a process known as no‐go decay (NGD) . The structure of Dom34‐Hbs1 complex is similar to that of eRF1 and eRF3, but Dom34 lacks the motifs for codon recognition and peptide release , . Along with studies from biochemical analyses, it is proposed that the Dom34‐Hbs1 complex binds to the ribosomal A site to promote subsequent dissociation of ribosome subunits for ribosomal recycle , . The molecular connection between Dom34‐Hbs1‐mediated ribosomal recycle and mRNA cleavage, and the identity of the responsible endonuclease remain unclear. In addition to the RNA quality control in the NGD pathway, Dom34‐Hbs1 is also important for non‐stop decay, decay of non‐functional 18S rRNAs and mRNAs with premature stop codon , , . Therefore, the Dom34‐Hbs1 complex appears to represent a major RNA surveillance pathway in eukaryotes to detect and rescue the ribosomes stalled on mRNAs during translation.
Here, we report that the Drosophila Pelo is required for silencing of germline transposons in the ovary and testis. Pelo is not required for piRNA biogenesis or function, and our analyses indicate that Pelo might function together with Hbs1, possibly via a NGD‐like mechanism, to prevent translation of transposon mRNAs.
Results and Discussion
To facilitate the study on the potential role of Pelo in transposon silencing, we generated a few hypomorphic alleles of pelo by imprecise excision of P‐elements inserted in the pelo locus (Fig EV1). Based on molecular lesion, gene expression abundance, and ovary phenotype, we considered peloPB60 as the strongest loss‐of‐function allele, peloPA13 the weakest, and pelo1 in‐between (see Materials and Methods, and Fig EV1). By real‐time PCR analysis, we observed moderate but significant up‐regulation of selective germline transposons in ovaries of pelo homozygous or transheterozygous mutants (Fig 1A). The magnitude of TE up‐regulation was correlated with the strength of pelo mutations (Fig 1A) and was further enhanced by ambient temperature (Fig EV2). In addition, up‐regulation of multiple TE elements, such as HeT‐A, 1731, and I‐element, found in peloPB60 homozygous ovaries could be significantly suppressed by germline expression of a pelo transgene (Fig 1B). Using antibodies to I‐element ORF1, we also observed protein accumulation in pelo ovaries in the oocyte of the stage 2 egg chambers in all ovarioles examined (23/23). As controls, accumulation of I‐element protein was only observed in the oocyte of developing egg chambers in aub mutant ovaries, but not in wild‐type ovaries (Fig 1C). Taken together, these observations suggest that pelo is required for transposon silencing in the Drosophila ovary whose mutation causes up‐regulation of some germline TEs at both mRNA and protein levels.
In Drosophila testis, the repeated Stellate (Ste) element is normally silenced by the piRNA pathway mediated by Su(Ste) piRNAs . As expected, in aub mutant testis, where the piRNA pathway is compromised, Ste proteins accumulated and formed needle‐like crystals in the spermatocyte (Fig 1D). Germline knockdown of pelo by nos‐Gal4/UAS‐pelo‐RNAi also led to Ste crystal formation in the spermatocytes with 100% penetrance (49/49) though the severity varied in different testis (Fig 1D). Strong loss‐of‐function mutants of pelo (pelo1/PB60) did not display the crystal phenotype in spermatocytes (Fig 1D), possibly because the mutant spermatocytes have not yet developed to a later stage necessary for Ste expression, as Pelo is required for the progression through meiosis I during spermatogenesis . Consistent with this notion, leaky expression of a UAS‐pelo transgene (without a GAL4 driver) in pelo1/PB60 mutants also caused Ste crystals in the spermatocytes (Fig 1D). We generated another pelo transgene with a GFP tag to its C‐terminal and found that this transgene is only partially functional because the transgene allowed the formation of spermatids, but failed to rescue the sterile phenotype of pelo mutant males (data not shown). Germline expression of the pelo‐GFP transgene in pelo null testis also led to the formation of Ste crystals (Fig 1D). These data suggest that Pelo is required for Ste silencing in the developing spermatocytes.
The Ste elements are distributed in two clusters in the genome, the 12D euchromatic region and the distal X‐heterochromatic region. Similar to previous observations, Ste transcripts derived from either locus was significantly up‐regulated (approximately 25‐fold) in AubQC42/HN2 mutant testes  (Fig 1E). By contrast, only transcripts derived from the euchromatic locus showed significant up‐regulation in pelo‐RNAi testis, and the magnitude of mRNA up‐regulation was also comparatively less dramatic (approximately fourfold) (Fig 1F).
Pelo could regulate TE silencing through the piRNA pathway or other mechanisms. We first determined whether Pelo regulates the expression or function of genes in the piRNA pathway. The mRNA levels of virtually all major known genes involved in the piRNA remained unchanged in pelo mutant ovaries, except for Yb, whose expression was up‐regulated (Fig 2A). The protein levels for several major piRNA pathway components, such as Piwi, Vasa, and Krimp, remained unaltered as well (Fig 2B). In addition, the subcellular distribution patterns of all piRNA proteins examined, including Piwi, Aub, Ago3, Vasa, and Krimp, remained unaltered in pelo mutant germline cysts (Fig 2C). The above data suggest that Pelo does not regulate the expression or subcellular localization of major protein components involved in the piRNA pathway.
To determine whether Pelo affects piRNA biogenesis, we profiled all small RNAs extracted from pelo1/+ and pelo1/PB60 ovaries by deep sequencing. After filtering out tRNA, rRNA, and degraded snoRNAs, we had about 10M reads for each sample, 60% of which mapped to the repetitive DNA regions in the genome (Fig EV3). The RNA profile in general showed that the production of endogenous siRNA (esiRNA), piRNAs, and microRNAs (miRNAs) in pelo1/PB60 ovaries was largely similar to that in the control ovaries. We specifically analyzed small repetitive RNAs uniquely mapped to the two major piRNA clusters, the 240‐kb 42AB cluster that exclusively processes germline piRNAs, and the 179‐kb flamenco cluster that predominantly produces somatic piRNAs. Both the abundance and distribution of small RNAs derived from these two clusters were virtually unchanged in pelo1/PB60 ovaries (Fig 3A). We further examined the small RNAs mapped to specific transposable DNA elements. HeT‐A is a germline‐specific TE, and ZAM is soma‐specific. The abundance of small RNAs derived from both TEs also remained unaltered in pelo1/PB60 ovaries (Fig 3B). In parallel, we examined the production levels of several piRNAs, including roo, HeT‐A, and AT‐chX‐1, by polyacrylamide gel electrophoresis and Northern blotting analysis. As a positive control, piRNAs were significantly reduced in AubQC42/HN2 ovaries (Fig 3C). We found that the production of these piRNAs remained unchanged in pelo1/PB60 ovaries, and the production of miR‐8 was unaltered in either AubQC42/HN2 or pelo1/PB60 ovaries (Fig 3D). Finally, Northern blot analysis revealed that Su(Ste)‐4 piRNA production in testis was significantly compromised in Aub mutants but remained at comparable level to the control in pelo‐RNAi mutants (Fig 3E). Taken together, these data agree with the global RNA profiling analysis and suggest that Pelo does not regulate the biogenesis of small RNAs, including piRNAs and miRNAs. In addition, Pelo does not seem to generally regulate the levels of mRNAs. As shown previously, the mRNA expression of many germline‐specific genes was largely unaltered in pelo mutants (Fig 2A). We also examined a set of housekeeping genes (polymerase genes), and their mRNA expression was virtually unaltered or within twofold of up‐regulation in pelo mutants (Fig EV3).
In yeast, Dom34/Pelo complexes with Hbs1 in the NGD process to regulate endonucleolytic cleavage of mRNAs in stalled ribosomes during translational elongation . We found that in Drosophila, Pelo displayed abilities to interact with the Drosophila Hbs1 (CG1898) in the yeast two‐hybrid assay (Fig 4A). Furthermore, Pelo co‐immunoprecipitated with Hbs1 in ovarian extracts (Fig 4B). To determine whether Pelo functions in a complex with Hbs1 in regulating TE silencing in the Drosophila germline, we generated a Hbs1 null Drosophila strain. Ovaries from Hbs1 mutants showed a moderate but significant increase in TE expression levels compared to the wild‐type controls (Fig 4C). In addition, mutation of Hbs1 significantly enhanced the up‐regulation of HeT‐A element expression caused by pelo mutation, although it did not further increase 1731 or ZAM expression (Fig 4D). In yeast NGD pathway, mutational analysis suggests that Hbs1 has a much minor role in the process compared to the requirement for Dom34, possibly due to the redundant functions by other eRF3‐like proteins . Structural and biochemical data have shown that the conserved PGF motif in the central domain of Pelo/Dom34 interacts with a conserved RDF motif in the GTPase domain of Hbs1. The PGF motif is also required for efficient NGD activity as a P to A mutation in this motif, which does not completely abolish the interaction , reduces NGD activity , . We therefore generated a transgene for Pelo carrying P210 to A (P210A) mutation in the PGF motif and examined the ability of the mutant Pelo in rescuing the observed defects in pelo1/PB60 ovaries. Indeed, the P210A mutant Pelo had significantly reduced ability to repress TE levels (Fig 4E). These observations suggest that Pelo might form a complex with Hbs1 (and possibly other eRF3‐like proteins) in the Drosophila germline to regulate transposon silencing.
In yeast, RpS30a, a ribosomal protein, has been identified as a high‐copy suppressor of the growth defect caused by Dom34 ablation . Interestingly, overexpression of RpS30a is able to complement the NGD defects and allow some cleavage of NGD substrates in a Dom34 strain . The underlying mechanism is unclear, but it has been speculated that extra RpS30a might extend the elongation pausing allowed for mRNA cleavage . We generated a transgenic fly expressing the Drosophila RpS30a and found that RpS30a overexpression in the germline significantly reduced the TE levels in pelo−/− ovaries, an effect that is largely similar to overexpression of Pelo (Fig 4F). By contrast, overexpression of RpS30a failed to reduce the TE levels in AubQC42/HN2 ovaries (Fig 4G). Germline overexpression of RpS30a also improved the egg production (Fig 4H) and the hatching rate of pelo females, similar to the germline expression of Pelo (Fig 4I). Taken together, these data indicate that Pelo might regulate transposon silencing at the translational level, possibly through the NGD pathway to cleave TE transcripts that have been loaded with ribosomes, thereby preventing translation.
Studies with artificially expressed mRNAs have shown that the Dom34‐Hbs1 complex can detect and dissociate stalled ribosomes with no‐stop mRNAs . But the in vivo targets are largely unknown. A recent study also suggests that Dom34 does not generally dissociate ribosomes on coding sequence but on 3′ UTRs . Because TE transcripts loaded on the ribosomes could still be potentially recognized by complementary piRNAs, one possibility is that the binding of piRNAs to the transcript may produce no‐go mRNAs, followed by endonucleolytic cleavage via NGD. If this is true, Pelo‐mediated TE silencing must rely on proper piRNA biogenesis. In Ago3‐ and Aub‐depleted ovaries, where the piRNA biogenesis is reduced, we found that mutation in pelo was able to further enhance Het‐A and 1731 up‐regulation (Fig EV4). In addition, depletion of Pelo could still further enhance TE up‐regulation by additionally depleting Dcr2 (Fig EV4), which generates esiRNAs that are implicated in TE silencing in somatic cells , . Because disrupting the ping‐pong cycle may reduce, but not eliminate the piRNA production, it remains undetermined whether Pelo‐mediated TE silencing is dependent or independent of piRNAs.
RNA surveillance mediated by Dom34‐Hbs1 appears to be an evolutionarily conserved mechanism from yeast to human , . We found that expression of the mouse Pelo (mPelo) gene in Drosophila was able to rescue the observed defects found in pelo1/PB60 mutants, including TE depression (Fig 5A) and defects in spermatogenesis (Fig 5B and C). Ubiquitous expression but not germline‐restricted expression of mPelo effectively rescued the sterility of pelo1/PB60 mutants (Fig 5D), suggesting that the function of Pelo in transposon silencing could be conserved in mammals. On another note, insufficient rescue by the germline‐restricted expression of Pelo indicates a role for Pelo in somatic cells as well.
Our studies here suggest an interesting hypothesis that in addition to the transcriptional and posttranscriptional levels of TE silencing by Piwi proteins and piRNAs, the mRNA surveillance complex Pelo‐Hbs1 may provide another level of defense against TE transcripts that have escaped from the piRNA pathway‐mediated endonucleolytic cleavage. One plausible mechanism would be that these escaped transcripts are loaded with ribosomes for translation, but the NGD machinery is able to recognize them, followed by degradation. How the loaded TE mRNAs are recognized by the NGD machinery is unclear, but the involvement of piRNAs remains to be a possibility worthy of future investigation. Given that TEs are particularly active in the germline, the involvement of multiple levels of defense against TEs in the germline ensures the integrity of genetic information to be passed to the next generation. We propose that the Pelo‐Hbs1 RNA surveillance complex as another gatekeeper against the expression of transposons could be a common feature in the metazoan germline.
Materials and Methods
Additional methods are described in the Appendix file.
Flies were cultured on standard food media with yeast paste added to the food surface. The culture temperature was 25°C unless otherwise noted. Strains used in this study were as follows: Ago3T2, Ago3T3, AubQC42, and AubHN2 (gifts from Phillip Zamore); pelo1 is a P‐element insertional allele , and RT–PCR analysis suggests that this allele produces a C‐terminal truncated product. peloPB60 and peloPA13 are insertion and deletion alleles, respectively, generated by imprecise excision of P‐element insertions: peloPB60 has a 107‐base‐pair deletion in the 2nd exon which creates an early stop codon and can be considered as a genetic null allele; peloPA13 has a 558‐base‐pair insertion in the 5′ UTR, and RT–PCR analysis suggests that the mutation causes approximately 80% reduction of gene product (see Fig EV1); Hbs1109 (a null allele, details will be described elsewhere); nos‐Gal4VP16 ; UAS‐pelo‐RNAi (VDRC #34770, #34771); and tub‐Gal4 (BDSC #5138).
Generation of transgenic flies
To make a UASp‐RpS30a construct, the RpS30a cDNA was cloned from a w1118 ovarian cDNA library and then subcloned into a pUASP vector with KpnI and XbaI. Mouse pelo cDNA from C57BL/6 mice (The Jackson Laboratory) was cloned into a pUASp vector with KpnI and XbaI. The Gateway cloning technology (Invitrogen) was used to generate UASp‐pelo‐GFP, UASp‐pelo‐Flag, and UASp‐Myc‐Hbs1 constructs. The P210A point mutant was generated by QuickChange Site‐Directed Mutagenesis Kit (Stratagene). All the plasmids were verified by DNA sequencing. The plasmid DNA was introduced to w1118 embryo by a standard procedure to generate transgenic flies.
Immunostaining and microscopy
Drosophila ovaries were dissected and immunostained as described previously . The following primary antibodies were used: anti‐Piwi, anti‐Ago3, anti‐Aub (1:1,000, gifts from Gregory J. Hannon), anti‐Krimp (1:10,000, a gift from Toshie Kai), anti‐Vasa (DSHB, 1:100), anti‐Stellate (1:1,000, a gift from William E. Theurkauf), anti‐I‐element ORF1p (1:50, a gift from David Finnegan), anti‐β‐gal (DSHB, 1:50), and anti‐cleaved caspase‐3 (Cell Signaling, 1:300). For IC staining, fixed testes were incubated with rhodamine‐conjugated phalloidin (Molecular Probes, 1:10) at 37°C for 1 h. Secondary antibodies, including goat anti‐rabbit and goat anti‐mouse IgGs, conjugated to Alexa (488 or 568) (Molecular Probes) were used at a dilution of 1:300 (DAPI (4′,6‐diamidino‐2‐phenylindole), Sigma; 0.1 mg/ml, 5 min incubation). Images were collected by either a Zeiss Meta 510 confocal microscope system or a Zeiss Imager Z1 equipped with an ApoTome. All acquired images were processed in Adobe Photoshop and Illustrator.
RNA isolation and qPCR assays
Total RNA from 10‐20 ovaries was extracted by TRIzol reagent (Invitrogen). After DNase treatment, complementary DNA (cDNA) was synthesized using an oligo dT primer and High‐Fidelity cDNA Synthesis Kit (Roche). RT–qPCR was performed in three duplicates using SYBR Premix Ex Taq RT‐PCR Kit (Takara) on an ABI PRISM 7500 fast Real‐time PCR System (Applied Biosystems). Endogenous Actin5c mRNA levels were measured for normalization. Fold changes for mRNA were calculated using ΔΔCt method . The primers used are listed in Table EV1.
Small RNA isolation and Northern blot
Small RNA was enriched by PEG8000 precipitation from total ovaries RNA, and Northern blot was performed according to Qi et al . 32P‐end‐labeled oligonucleotides complimentary to small RNA sequence were used as probes which are listed in Table EV1.
To test female fertility, two virgins were collected and mated with two 5‐ to 7‐day‐old w1118 males in a small cage with 35‐mm apple juice agar plate with yeast paste as described . After 2 days, the plate was replaced by a new one every day. The number of eggs per female per day was counted every 24 h, and the hatching rate was scored 48 h after the plate was changed. At least eight plates were scored in total for each genotype at each time point. To test male fertility, each 2‐ to 5‐day‐old male was collected and mated with three 3‐ to 7‐day‐old w1118 virgins. Fertility was determined by examining the appearance of larvae 5 days after mating.
Small RNA cloning and sequencing
Ten micrograms enriched small RNA were separated on a 15% denaturing polyacrylamide gel.
18‐ to 30‐nt RNAs were purified according to RNA oligo markers and ligated to adapters to generate the libraries for subsequent sequencing on the Illumina GA II instrument. The small RNA‐seq library was prepared by following the manufacturer's instructions.
The GEO accession code for the RNA‐seq data is GSE69468.
Data are presented as mean ± SEM. P‐values were calculated using one‐way ANOVA or unpaired two‐tailed Student's t‐test by GraphPad Prism 5 (GraphPad Software Inc.).
FY, RZ and RX conceived and designed the experiments, FY and RX analyzed the data and wrote the manuscript. FY, RZ, XF, HH, YX, YM, HC, TC, and YQ performed the experiments and analysis.
Conflict of interest
The authors declare that they have no conflict of interest.
Expanded View Figures PDF
We thank Phillip Zamore, Greg Hannon, David Finnegan, Toshi Kai, William Theurkauf, the Bloomington Drosophila Stock Center, and Vienna Drosophila Resource Center for providing fly stocks and reagents; Xijuan Li and Anying Kang for technical assistance; and members of the Xi Laboratory for helpful comments and discussion. This work was supported by National Basic Science 973 grants (2011CB812700 and 2014CB849700) from the Chinese Ministry of Science and Technology.
FundingNational Basic Science2011CB8127002014CB849700
- © 2015 The Authors