Evidence for maternally transmitted small interfering RNA in the repression of transposition in Drosophila virilis

Abstract

Hybrid dysgenesis in Drosophila is a syndrome of gonadal atrophy, sterility, and male recombination, and it occurs in the progeny of crosses between males that harbor certain transposable elements (TEs) and females that lack them. Known examples of hybrid dysgenesis in Drosophila melanogaster result from mobilization of individual families of TEs, such as the P element, the I element, or hobo. An example of hybrid dysgenesis in Drosophila virilis is unique in that multiple, unrelated families of TEs become mobilized, but a TE designated Penelope appears to play a major role. In all known examples of hybrid dysgenesis, the paternal germ line transmits the TEs in an active state, whereas the female germ line maintains repression of the TEs. The mechanism of maternal maintenance of repression is not known. Recent evidence suggests that the molecular machinery of RNA interference may function as an important host defense against TEs. This protection is mediated by the action of endogenous small interfering RNAs (siRNAs) composed of dsRNA molecules of 21-25 nt that can target complementary transcripts for destruction. In this paper, we demonstrate that endogenous siRNA derived from the Penelope element is maternally loaded in embryos through the female germ line in D. virilis. We also present evidence that the maternal inheritance of these endogenous siRNAs may contribute to maternal repression of Penelope.

Fig. 1.

Putative Penelope siRNA is present in whole adults of strain 160, absent from strain 9, and maternally inherited in the nondysgenic cross. (A) Sequence organization of Penelope and location of the probe for siRNA. Black arrows indicate terminal repeats. RT, reverse transcriptase domain. (B) Northern analysis with 50 μg of total RNA from adults of strain 9 and 160, as well as dysgenic and nondysgenic progeny and sense and antisense orientations of the Penelope probe. 5S RNA is shown as a loading control, with a 10-bp RNA ladder shown on the side. Above the blots, we designate chromosomes as bars, with chromosomes from strain 160 in black and chromosomes from strain 9 in white. The pair of sex chromosomes, with the Y chromosome designated with a hooked bar, is shown above, and a representative pair of autosomes is shown below. Maternal cytoplasm is designated with an oval, with white and black indicating the presence or absence, respectively, of strain 160 chromosomes in the mother. Sense and antisense Penelope RNA between 20 and 30 nt is absent from strain 9 and present in strain 160. In the progeny, putative siRNA is present in males and females of the dysgenic (Dys) cross but only present in females of the nondysgenic (Ndys) crosses. (C) Northern analysis of 5 μg of enriched RNA from 0- to 2-h, nondysgenic and dysgenic embryos with 5S loading control. DNA oligos of 20, 24, and 28 nt with antisense and sense orientation to the Penelope probe were used as a polarity control. Small sense and antisense Penelope RNA is detectable in early embryos of the nondysgenic cross but not in those of the dysgenic cross. (D) RT-PCR for ftz expression in 0- to 2-h and 0- to 13-h nondysgenic embryos. Reverse transcriptase-negative controls (-) and rp49-positive controls (+) are shown. Lack of ftz expression in 0- to 2-h embryos indicates that genome-wide transcription has not yet been initiated in the nondysgenic embryos from C.

Fig. 2.

Putative siRNA is driven from the X chromosome of strain 160. (A) Northern analysis of 5 μg of enriched RNA per lane (with 5S loading control) shown for dysgenic (Dys) and nondysgenic (Ndys) adults and for adult progeny of the XX̂ and X9 crosses. Membrane was stripped and reprobed. Expression of Penelope siRNA in adults correlates with the presence of the X chromosome from strain 160. (B) Northern analysis of total RNA from the progeny of a backcross (dysgenic females × 9 males, lane labeled BC) containing a random mixture of recombinant X chromosomes (shown with stipled bars). Penelope siRNA is found in males and females. Cytoplasm in backcross adults is designated black, indicating the presence of 160 chromosomes in the mother.

Fig. 3.

The X chromosome is the source of Penelope siRNA within the female germ line. In 0- to 2-h embryos, Penelope siRNA is found only if mothers possess the 160 X chromosome. In females, the X chromosome drives the expression of Penelope siRNA in the germ line and soma. Females that lack this chromosome do not demonstrate Penelope siRNA in either tissue. Five micrograms of enriched RNA was used per lane.

Fig. 4.

The contribution of maternal 160 chromosomes to the repression of gonadal atrophy. (A) Wild-type testis (left) and atrophied testis (right). (B) Genetically identical progeny of crosses with or without an attached-X chromosome (XX̂) provide evidence that hybrid dysgenesis in males results from lack of maternally transmitted repression. Shown is the estimated probability of gonadal atrophy in genetically identical males that differ only in respect to the cytoplasm of the mother. Males that possess the X chromosome from strain 160 but whose mothers lack chromosomes from strain 160 show high levels of gonadal atrophy (n = 211). Males whose mothers possess 160 chromosomes do not (n = 228). (C) Maternal effects of individual strain 160 chromosomes on suppression of gonadal dysgenesis; the lower the bar, the greater the level of repression. The largest effects are due to the X chromosome and chromosome V.