Production of Small Noncoding RNAs from the flamenco Locus Is Regulated by the gypsy Retrotransposon of Drosophila melanogaster

Abstract

Protective mechanisms based on RNA silencing directed against the propagation of transposable elements are highly conserved in eukaryotes. The control of transposable elements is mediated by small noncoding RNAs, which derive from transposon-rich heterochromatic regions that function as small RNA-generating loci. These clusters are transcribed and the precursor transcripts are processed to generate Piwi-interacting RNAs (piRNAs) and endogenous small interfering RNAs (endo-siRNAs), which silence transposable elements in gonads and somatic tissues. The flamenco locus is a Drosophila melanogaster small RNA cluster that controls gypsy and other transposable elements, and has played an important role in understanding how small noncoding RNAs repress transposable elements. In this study, we describe a cosuppression mechanism triggered by new euchromatic gypsy insertions in genetic backgrounds carrying flamenco alleles defective in gypsy suppression. We found that the silencing of gypsy is accompanied by the silencing of other transposons regulated by flamenco, and of specific flamenco sequences from which small RNAs against gypsy originate. This cosuppression mechanism seems to depend on a post-transcriptional regulation that involves both endo-siRNA and piRNA pathways and is associated with the occurrence of developmental defects. In conclusion, we propose that new gypsy euchromatic insertions trigger a post-transcriptional silencing of gypsy sense and antisense sequences, which modifies the flamenco activity. This cosuppression mechanism interferes with some developmental processes, presumably by influencing the expression of specific genes.

Keywords: RNA silencing; ecdysis; primary transcript; small RNA; transposon.

Figure 1

Lethal ecdysis deficiency is induced by a gypsy insertion in the cut locus. (A) Cuticle preparations of wings from flam^A/ flam^A (+/+), ct^A flam^A/ct^A flam^A (ct^A/ct^A), ct^A flam^A/ct⁶ (ct^A/ct⁶), and ct^A flam^A/ct^A flam^A; su(Hw)⁸/su(Hw)⁸ (ct^A/ct^A; su(Hw)⁸/su(Hw)⁸). (B, C) Mortality during adult eclosion of flies in the AstC-R2 ^f03116 background. (B) Mortality during adult eclosion of heterozygous and homozygous flies derived from the mating of AstC-R2 ^f03116 heterozygous parents. (C) Mortality during adult eclosion of ct^A heterozygotes and homozygotes, and AstC-R2 heterozygous and homozygous flies derived from mating of AstC-R2 heterozygous parents. (D, E) representative pictures of ct^A/ct^A; AstC-R2 ^f03116/AstC-R2 ^f03116 pharate adults. (D) Fly trapped inside the puparium ∼12 hr after the start of the adult ecdysis, showing the characteristic pigmentation of eclosed adults. (E) Fly extracted from puparium showing pupal membrane attached to legs, wings, and other parts of the body (arrow). (F) Mortality during adult eclosion of flies with the third chromosomes deriving from the y w^67c23 strain (indicated in brackets) in absence or presence of the ct^A mutation and of the TM6b balancer chromosome.

Figure 2

Genetic interactions between flamenco permissive alleles and gypsy insertions induce specific phenotypes. (A–E) Representative pictures of egg chambers subjected to β-galactosidase staining as readout for gypsy-lacZ reporter activity. (A) gypsy-lacZ reporter activity is repressed when the flam^su(f) restrictive allele is combined with the Df(1)l11 deficiency encompassing the flamenco locus. (B) When the same deficiency is combined with the X chromosome currying the flam^A allele, gypsy-lacZ reporter activity is strongly induced. (C) gypsy-lacZ reporter activity is repressed when the flam^FM7c restrictive allele is combined with the flam^A allele. (D) gypsy-lacZ reporter activity is induced in flam^A/flam¹ ovaries. (E) Combination of the Df(1)l11 deficiency with the ct^A allele in the flam^A-containing chromosome significantly reduces β-galactosidase staining. (F) Mortality during adult eclosion of ct^A flam^BG flies compared to the flam^BG strain. (G) Mortality during adult eclosion of ct⁶ and ctⁿ flies in their original genetic background and in combination with flam¹. (H) Mortality during adult eclosion of flam^A/flam¹ and ct^A flam^A/ct⁶ flam¹ female (F) flies (** P < 0.01). (I, J) Wings derived from male flies hemizygous for Bx² allele in (I) its original genetic background and (J) in flam¹ background. (K) Frequency of wing-blister phenotype of Bx² wings in their original genetic background and in combination with flam¹.

Figure 3

The gypsy insertion in the cut locus induces changes in the expression of gypsy and flamenco. (A) Map of the flamenco cluster showing the DIP1 gene and the flam^BG02658 (flam^BG) insertion at the 5′ end of the locus, the gypsy transposon fragments (thick bars), the positions of primers used to analyze flamenco primary transcripts expression (numbers 1–6), and the position of tested piRNAs (letters A and B). (B–E) qRT-PCR analysis of gypsy, ZAM, Idefix, and specific flamenco regions in RNA isolated from (B) 48-hr-old pupae, (C) 96-hr-old pupae, (D) 0- to 24-hr-old female adult heads, and (E) 3-day-old adult ovaries. Shown are average levels (n = 3), and error bars indicate SD (* P < 0.05, ** P < 0.01, *** P < 0.005).

Figure 4

Lethal ecdysis deficiency and flamenco modulation induced by gypsy seems to depend on post-transcriptional silencing. (A, B) Mortality during adult eclosion of ct^A flam^A flies carrying mutations affecting heterochromatin or small RNA pathways. (A) Analysis of mutations affecting heterochromatin formation. As an additional control, the heterozygous combination of a third chromosome from the yw^67c23 strain and a third chromosome from the Canton-S strain was also analyzed. The genotype of the flies from which the third wild-type chromosome originates is enclosed in brackets. (B) Analysis of mutations affecting endo-siRNA and piRNA production. (C–E) ChIP analysis. Cross-linked chromatin of adult heads from w flam^A and w ct^A flam^A females was IP with antibodies specific to (C) H3K9me3, (D) H3K27me3, and (E) no antibody as negative control. The IP DNA was analyzed by qPCR. Protein binding is expressed as the percentage of input and is shown for each primer set. cut-up and cut-down primers amplify upstream and downstream of the gypsy insertion at the cut locus, respectively. TART is used as a positive region for H3K9me3, and rp49 as a negative region showing background levels. Shown are average levels from two independent experiments and error bars indicate average deviation.

Figure 5

Cosuppression of gypsy and flamenco sequences from which small RNAs against gypsy originate. qRT-PCR analysis. Shown are the transcript levels of gypsy and two gypsy fragments in the flamenco locus (primers 5–6, described in Figure 3A) in RNA isolated from 48-hr-old pupae carrying the homozygous (A) ct^A or (B) f^A mutation induced by gypsy. (C) Expression of gypsy and gypsy fragments inside flamenco in 0- to 24-hr-old adult heads from flies carrying the ct^A homozygous mutation. (D) Expression of the distal gypsy fragment (primers 6) in w flam^A adult ovaries compared to that in 48-hr-old pupae. (E) piRNA levels in 3-day-old adult ovary detected by TaqMan specific probes (see Materials and Methods for details). Position of analyzed piRNAs loci are indicated in Figure 3A. Shown are average levels (n = 3), and error bars indicate SD (* P < 0.05, ** P < 0.01, *** P < 0.005).

Figure 6

The gypsy insertion in the cut locus triggers the expression of fused transcripts from flanking genomic regions into the gypsy sequences. (A) Map of the gypsy insertion site in the cut locus and of the primers used in RT-PCR experiments (small arrows). Stand specific RT primers are represented in grey while qPCR primers are represented in black. fw arrow indicates direction of telomere-to-centromere expression while re arrow indicates direction of centromere-to-telomere expression. (B–F) RT-PCR experiments performed using RNA isolated from 0- to 24-hr-old female heads. (B, C) Strand-specific qRT-PCR reveals transcription in the genomic regions surrounding the gypsy insertion site in the cut locus. Transcription levels are variable and influenced by the pattern of the gypsy mutations present in each strain. Shown are average levels (n = 3), and error bars indicate SD. Retrotranscription of the cut-up site was primed by (B) primers F-RT up and R-RT up, while retrotranscription of the cut-down site was primed by (C) primers F-RT dw and R-RT dw. Primers cut-up were used to amplify the cut-up region while primers cut-dw were used to amplify the cut-down region. (D) Strand-specific RT-PCR products loaded in a 2% agarose gel confirming the presence of fused transcripts only in flies carrying the gypsy insertion in the cut locus. Primers cut-gyp up were used to amplify the forward fused transcript while primers cut-gyp dw were used to amplify the reverse fused transcript. DNA ladder scale is in base pairs. (E) Strand-specific qRT-PCR analysis of the transcription levels of the regions cut-gyp up and cut-gyp dw. Shown are average levels (n = 3), and error bars indicate SD (* P < 0.01). (F) qRT-PCR analysis. Shown are the transcript levels of gypsy and of three regions inside the flamenco locus (primers 1, 5, and 6; described in Figure 3A). Shown are average levels (n = 3), and error bars indicate SD (* P < 0.05).