Adaptive regulation of testis gene expression and control of male fertility by the Drosophila hairpin RNA pathway. [Corrected]

Abstract

Although endogenous siRNAs (endo-siRNAs) have been described in many species, still little is known about their endogenous utility. Here, we show that Drosophila hairpin RNAs (hpRNAs) generate an endo-siRNA class with predominant expression in testes. Although hpRNAs are universally recently evolved, we identify highly complementary protein-coding targets for all hpRNAs. Importantly, we find broad evidence for evolutionary divergences that preferentially maintain compensatory pairing between hpRNAs and targets, serving as first evidence for adaptive selection for siRNA-mediated target regulation in metazoans. We demonstrate organismal impact of hpRNA activity, since knockout of hpRNA1 derepresses its target ATP synthase-β in testes and compromises spermatogenesis and male fertility. Moreover, we reveal surprising male-specific impact of RNAi factors on germ cell development and fertility, consistent with testis-directed function of the hpRNA pathway. Finally, the collected hpRNA loci chronicle an evolutionary timeline that reflects their origins from prospective target genes, mirroring a strategy described for plant miRNAs.

Figure 1. hpRNAs Are Biased to Testis and Processed by the RNAi Pathway

(A) Small RNA levels in reads per million (RPM). The top row shows hpRNA loci. While some of these generate reads in multiple libraries, all are predominantly expressed in testes and in mass-isolated imaginal disc libraries that contain larval gonads. (B) qPCR analysis of pri-hpRNA1 confirms much higher levels in testis than in whole adults or in dissected imaginal discs that are free of gonadal contamination. (C) Other small RNA classes, including miRNAs and siRNAs derived from TEs and cis-NATs, do not exhibit the class-wide testis bias of hpRNAs. (D) Analysis of small RNA libraries from loqs-PD mutant males (upper) and dcr-2 mutant males (lower) show that all hpRNA-derived siRNAs are strongly downregulated in both conditions, as are TE-siRNAs and cis-NAT-siRNAs. The “long” duplex canonical miRNAs mir-989 and mir-956, whose lengths overlap those of the shortest hpRNAs (e.g., hpRNA1) were not downregulated. (E) Northern blotting of hpRNA expression constructs transfected into S2 cells demonstrates that hpRNAs can be functionally defined as siRNAs, since their bulk population in total RNA is resistant to β-elimination while the bantam miRNA is sensitive. Moreover, their resultant small RNAs are preferentially sorted to AGO2, while the bantam miRNA is preferentially sorted to AGO1. (F) Luciferase sensor tests confirm regulatory capacity of transfected hp-mir-997 and hpRNA1 expression constructs. Data are the mean of quadruplicate assays ± SEM. See also Figures S1–S3 and Tables S1 and S2.

Figure 2. Drosophila hpRNA1 Is an Endogenous Repressor of ATP-Synthase-β

(A) Drosophila hpRNA1 exhibits extensive complementarity to ATP-synthase-β. The most highly expressed siRNA from the 5′ arm of hpRNA1 (5pA) guides the endogenous cleavage of ATP-synthase-β, as evidenced by the dominant isolation of 5′ RACE clones that terminate between nucleotides 10 and 11 of hpRNA1–5pA. The secondary RACE product was not related to any major hpRNA1 product. (B) RNA-seq data shows that ATP-synthase-β is expressed at lower levels in the testes relative to the rest of the animal. (C) qPCR validation of lower ATP-synthase-β in testes. (D) ATP-synthase-β is upregulated in dcr-2 mutant testis. Data are the mean of triplicate assays ± SEM.

Figure 3. Three hpRNAs of the pncr009 Family Target Multiple Neighboring Genes

(A) A 60 kb genomic region encompasses three related hpRNAs: hp-pncr009, hp-CR32207, and hp-CR32205 (red). These hpRNAs exhibit extensive complementarity to ten related protein-coding genes of the 825-Oak family (blue). Some of the hpRNAs are transcribed antisense to 825-Oak genes, whereas other 825-Oak genes form divergent pairs (gray highlighted). Small RNA sequencing evidence shows that the abundant siRNAs (dominant 21 to 22 nt reads) map to the hpRNA loci. (B) Zoomed view of hp-pncr009 and its antisense target CG32212. (C) Example of an siRNA that is commonly produced from two hpRNAs, which exhibits full complementarity to six different 825-Oak genes. (D) Phylogenetic tree shows that the left arms of all three hpRNAs cluster together with the mRNA targets, while their right arms form a separate clade. (E) Luciferase sensor assays demonstrate that multiple pncr009 hairpins can repress multiple genes of the 825-Oak family. Data are the mean of quadruplicate assays ± SEM. See also Figures S1 and S4.

Figure 4. Abundant Evidence for Compensatory Substitutions that Maintain Pairing of hpRNA Arms and of hpRNAs to their Targets

(A–C) Multiple alignments with compensatory substitution changes between the siRNA:passenger and siRNA:target are shown for (A) hpRNA1, (B) hp-CR32207, hp-CR32205, hp-pncr009, and (C) hp-mir-997. The highlighted compensatory substitution changes are relative to the reference species (dm). Three-way compensatory substitution is defined as (1) both siRNA:target and siRNA:passenger undergo double substitutions on the same base (dark green) or (2) siRNA:target undergoes a double substitution (dark green) and siRNA:passenger undergoes a single substitution on the same base (dark blue). Color legend for substitutions in the multiple alignments is shown on the right. Yellow bar highlights the columns where siRNA compensatory changes show adaptation to target change. Aside from the three-way compensatory changes highlighted by yellow bars, there are also cases where siRNA:target undergoes a double substitution and siRNA:passenger undergoes a double or single substitution on the same base, but it involves an unpaired base in the reference species. We do not consider such cases as strong three-way compensatory changes, but they do provide supporting evidence for maintenance of the structure and siRNA adaptation to target changes (highlighted by yellow bar in siRNA:target alignment only). See also Figure S1 and Tables S5.

Figure 5. An hpRNA1 Mutant Deregulates ATP synthase-β and Impairs Male Fertility, as Do RNAi Pathway Mutants

(A) A Minos element in CG4770 was mobilized to yield a deletion of two protein-coding genes and hpRNA1. We introduced an 8.6 kb genomic rescue fragment into this background, which covers the deleted loci and extends into both flanking genes. Alternatively, we introduced a similar genomic fragment in which hpRNA1 was deleted. We used the latter as a hpRNA1 mutant combination and the former as a wild-type comparison. (B) Loss of hpRNA1 causes upregulation of ATP synthase-β. (C) Male fertility assays. To exhaust their sperm capacity, we mated individual males to three females, serially transferred these every other day for 2 weeks, and counted all progeny (n > 50 males all genotypes). Hemizygous loss of hpRNA1 reduced male fertility, which was restored by the hpRNA1 genomic locus. Hemizygous null mutants of dcr-2 or ago2 also caused strong fertility defects, which were also rescued by cognate genomic trans-genes. Data are mean ± SEM. For each mutant condition, Wilcoxon rank-sum tests were done to compare them to [w1118] (gray) or to their cognate genomic rescue (blue); ***p < 0.0005. (D–G) Adult eyes that carry ey-Gal4 GMR-Gal4, and UAS-dcr-2 transgenes, with or without other responder transgenes. (D) The tester stock alone exhibits a normal eye. (E) Inclusion of UAS-hpRNA1 nearly obliterates the eye. (F) Coexpression of UAS-hpRNA1 and UAS-ATP-synthase β strongly rescues the eye. (G) Coexpression of UAS-hpRNA1 with a UAS-ATP-synthase β construct mutated for the hpRNA1-complementary region (see below) fully rescues the eye. See also Figure S6.

Figure 6. The hpRNA/RNAi Pathway Is Required for Spermatogenesis

Stainings of various regions of the male reproductive apparatus. Top two rows, seminal vesicle shown at low and high magnification; middle two rows, whole testis; bottom two rows, cystic bulges with individualization complexes. Scale bars are noted. (A and B) Wild-type seminal vesicle filled with needle-shaped sperm nuclei, as stained with DAPI. (C and D) hpRNA1 mutant seminal shows lower density of sperm, which is rescued by hpRNA1 locus ([E] and [F]). (G–I) Wild-type testis exhibits groups of individualization complexes with ordered nuclei and associated actin cones (arrowhead). These are mostly normally organized in hpRNA1 mutant (H) or rescued (I) testis. (J) High magnification of individual wild-type cystic bulge shows well-aligned ICs composed of actin (bracket). (K and L) (K)hpRNA1 mutant cystic bulge exhibits lagging ICs (asterisks), and this is fully rescuable (L). (M–T) Both null RNAi mutants, dcr-2 and ago2, are severely depleted for sperm in the seminal vesicle. (U and V) dcr-2 mutants exhibit highly dispersed spermatid nuclei. (W and X) ago2 mutants exhibit severely disorganized spermatid nuclei, such that it is difficult to identify individualization complexes. (Y and Z) dcr-2 mutants exhibit individualization complexes with reduced numbers and poorly aligned ICs ([Y’], bracket). About a third of cases exhibit even more dispersed ICs that are difficult to assign to cystic bulges. (AA) ago2 mutants exhibit highly dispersed ICs that do not organize into identifiable cystic bulges; this is rescued by ago2 transgene (BB). See also Figure S7.

Figure 7. Model for the Emergence and Evolutionary Maturation of Drosophila hpRNAs

(A) In an early stage of this process, two protein-coding genes in inverted orientation are subject to read-through transcription yielding a long inverted repeat. The resulting hairpin generates siRNAs that are self-complementary to the mRNAs. (A’) An alternate birth stage could be a protein-coding gene, which has an antisense hairpin-forming transcript, whose siRNAs are necessarily complementary. This arrangement is observed for each of the three pncr009 family hpRNAs, which are transcribed antisense to individual 825-Oak family members. (B) Subsequently, a distinct hairpin transcript may be retained in proximity to, and have extended complementarity to, the progenitor mRNAs. This state is reflected in the genomic clustering of pncr009 family hpRNAs and other targets of the 825-Oak family, many of which are still arranged in divergent orientations. (C) In the next step, the hpRNA and target become genomically separated yet retain extensive complementarity to each other. The hp-CG18854/CG8289 and hpRNA1/ATP synthase-β pairs are representative of this state. (D) Finally, in the mature state of this process, the hpRNA/target interaction has been whittled to a specific siRNA that is highly complementary to its target. This situation is exemplified by the hp-CG4068/mus308 interaction. (E) At various stages in the lifecycle, hpRNA amplifications are observed, either in cis or in distinct genomic copies. These additional copies may permit the aggregate loci to sample greater mutational space that facilitates target adaptation.