Expression of Drosophila virilis retroelements and role of small RNAs in their intrastrain transposition

Abstract

Transposition of two retroelements (Ulysses and Penelope) mobilized in the course of hybrid dysgenesis in Drosophila virilis has been investigated by in situ hybridization on polytene chromosomes in two D. virilis strains of different cytotypes routinely used to get dysgenic progeny. The analysis has been repeatedly performed over the last two decades, and has revealed transpositions of Penelope in one of the strains, while, in the other strain, the LTR-containing element Ulysses was found to be transpositionally active. The gypsy retroelement, which has been previously shown to be transpositionally inactive in D. virilis strains, was also included in the analysis. Whole mount is situ hybridization with the ovaries revealed different subcellular distribution of the transposable elements transcripts in the strains studied. Ulysses transpositions occur only in the strain where antisense piRNAs homologous to this TE are virtually absent and the ping-pong amplification loop apparently does not take place. On the other hand small RNAs homologous to Penelope found in the other strain, belong predominantly to the siRNA category (21nt), and consist of sense and antisense species observed in approximately equal proportion. The number of Penelope copies in the latter strain has significantly increased during the last decades, probably because Penelope-derived siRNAs are not maternally inherited, while the low level of Penelope-piRNAs, which are faithfully transmitted from mother to the embryo, is not sufficient to silence this element completely. Therefore, we speculate that intrastrain transposition of the three retroelements studied is controlled predominantly at the post-transcriptional level.

Figure 1. Transcription levels of selected D. virilis TEs.

(A) semiquantitative RT-PCR data for ovaries and carcasses; (B) Quantitative RT-PCR analysis of TE transcription levels in ovaries. Since RT-PCR failed to reveal any transcription of Penelope and Helena in strain 9, we do not include the results of comparative analysis of these TEs by qRT-PCR in the panel; (C) Northern blot detection of Ulysses and gypsyDv sense transcripts in strains 9 and 160. Poly-A RNAs isolated from strain 9, strain 160 and D. melanogaster yw^67c23 strain ovaries were used. The size of marker RNA is given in nt at the right. The filter was rehybridized with a fragment of constitutively expressed D. melanogaster rp49 to monitor the level of loaded RNA.

Figure 2. Whole-mount in situ RNA detection of sense transcripts of Penelope, Ulysses and gypsyDv in the ovaries of D. virilis strains 9 and 160.

(A) and (B) hybridization with Penelope-specific probe. No hybridization is seen in strain 9 (A), while in strain 160 (B) strong hybridization in the nurse cells cytoplasm is evident at stage 10. Ulysses-specific probe strongly hybridized with nurse cells nuclei in both strains at stages 2–10 (C, D, E). Arrows in E indicate putative RNA processing sites (foci), arrow-heads indicate putative nascent transcripts. Heavier label accumulation is usually observed in the cytoplasm of nurse cells of strain 9 (C). Reproducible hybridization of Ulysses probe with the centripetal (see arrow in F) and stretched follicle cells is a characteristic feature of strain 9 and 160 ovaries at stage 10. RNA in situ hybridization with gypsyDv-specific probe reveals hardly detectable labeling in the nurse cells cytoplasm in the ovaries of both strains studied (G–I). Significant hybridization of gypsyDv probe with follicle cells, which form appendages (H) and with follicle cells at the posterior end of ovarian chamber (I) represent the landmarks of strain 160.

Figure 3. The pattern of piRNAs distribution along transposons in testes.

Distribution of Ulysses-piRNAs in testes of strain 9 (A) and strain 160 (C). The distribution of piRNAs homologous to gypsyDv in testes of strain 9 (B) and strain 160 (D). Sense small RNAs are indicated in red, antisense – in blue.

Figure 4. Maternal deposition and distribution levels of Penelope-derived small RNAs.

siRNAs at (A, B) and piRNAs at (C, D) in strain 160 and its 0–2 h embryos. Sense small RNAs are indicated in red, antisense–in blue.

Figure 5. Frequency distribution of genomic mappings of Penelope–homologous si- and piRNAs.

Arrows indicate a proportion of Penelope-derived piRNA sequences mapping 2, 22 and 39 times in D. virilis genome.