A novel repeat-associated small interfering RNA-mediated silencing pathway downregulates complementary sense gypsy transcripts in somatic cells of the Drosophila ovary - PubMed (original) (raw)

A novel repeat-associated small interfering RNA-mediated silencing pathway downregulates complementary sense gypsy transcripts in somatic cells of the Drosophila ovary

Alain Pélisson et al. J Virol. 2007 Feb.

Abstract

Replication of the gypsy endogenous retrovirus involves contamination of the female germ line by adjacent somatic tissues. This is prevented by flam, an as-yet-uncloned heterochromatic pericentromeric locus, at the level of transcript accumulation in these somatic ovarian tissues. We tested the effect of a presumptive RNA silencing mechanism on the accumulation of RNAs produced by constructs containing various gypsy sequences and report that the efficiency of silencing is indeed correlated with the amount of complementary RNAs, 25 to 30 nucleotides in length, in the ovary. For instance, while these RNAs were found to display a three- to fivefold excess of the antisense strands, only the transcripts that contain the complementary sense gypsy sequences could be repressed, indicating that they are targeted at the RNA, not DNA, level. Their size and asymmetry in strand polarity are typical of the novel repeat-associated small interfering RNA (rasiRNA)-mediated pathway, recently suspected to prevent the deleterious expression of selfish DNA specifically in the germ line. Unlike microRNAs (but like rasiRNAs and, surprisingly, siRNAs as well), gypsy rasiRNAs are modified at the 3' end. The rasiRNA-associated protein Piwi (but not Aub) is required for gypsy silencing, whereas Dicer-2 (which makes siRNAs) is not. In contrast, piwi, aub, and flam do not appear to affect somatic siRNA-mediated silencing. The amount of gypsy rasiRNAs is genetically determined by the flam locus in a provirus copy number-independent manner and is triggered in the somatic tissues by some pericentromeric provirus(es), which are thereby able to protect the germ line from retroviral invasion.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

Schematic representation of the probes, constructs, and functional gypsy proviruses and transcripts. (A and B) Conserved structure of active gypsy proviruses and their genomic and subgenomic transcripts. The three ORFs (Gag, Pol, and Env) are indicated by light gray boxes, as are both 482-bp long terminal repeats (LTRs). The black and hatched boxes represent the small fragments inserted into the pES5 and pESpol constructs schematized below. The scheme is drawn to scale. (C) Sense and antisense RNA probes. The coordinates of the PCR fragments used to synthesize labeled RNA are from the gypsy sequence (GenBank accession number M12927). (D) Structure of the constructs. pES2 is a transcriptional fusion of the minimal ovarian yp3 promoter (dark gray box) with the prokaryotic lacZ reporter gene. It contains a unique XhoI restriction site in the 5′UTR that was used to insert small gypsy fragments in the indicated orientations, giving rise to the pES5, pESpol+, and pESpol− constructs.

FIG. 2.

FIG. 2.

Anatomy and biogenesis of rasiRNAs, miRNAs, and siRNAs. (A) The gypsy rasiRNAs fail to react with periodate, and their production does not require the Dicer-2 RNase III nor a high gypsy copy number. Low-molecular-weight-enriched RNA extracted from ovaries, submitted or not to NaIO4 treatment, was hybridized with a sense gypsy RNA probe (coordinates 6541 to 6718 in the gypsy sequence, accession number M12027) schematized in Fig. 1C. Hybridization of the membrane with the 310-433 sense riboprobe led to similar observations (data not shown). The names of the strains and/or genotypes used in this experiment are indicated above the corresponding lanes (see Materials and Methods for a description of the strains). The stripped membrane was reprobed with the end-labeled oligonucleotide complementary to the abundant 23-nt mir-13b miRNA (bottom panel). The amount of rasiRNAs in the MG1(rec) strain was found to be ∼0.8-fold that of the parental MG1 strain, after standardization with the miRNA loading control. A mixture of 5′-32P-radiolabeled 25- to 26-nt RNA oligonucleotides was added to the Decade kit (Ambion) to be used as size markers. At the top of the upper panel, the signal resulting from nonspecific hybridization with the abundant 30-nt 2S rRNA is the only one to be sensitive to the NaIO4 treatment. (B) Unlike miRNAs, the 21-nt siRNAs triggered by _Actin5C-Gal4-_driven expression of the UASt-IRlacZ hairpin in the somatic ovarian tissues are resistant to the periodate treatment. Low-molecular-weight enriched RNA extracted from ovaries, subjected or not to NaIO4 treatment, was hybridized with an antisense lacZ RNA probe (top panel) and reprobed with mir-13b (bottom panel). (C) In vivo expression of the GMR-wIR hairpin in the eye produce 21-nt siRNAs of both polarities that fail to react with periodate. Shown are two quantitative Northern blots of mirVana-extracted fly head RNA (Ambion). They were hybridized with either strand (s, sense; as, antisense) of a white exon 3 RNA probe. Proper standardization of both probes was achieved by cohybridization with dsRNA duplicate dot blots (not shown; see Materials and Methods). The bottom panels correspond to the simultaneous hybridization of both stripped membranes with the same mir-13b oligonucleotide probe. Different amounts of markers were loaded in lanes 5 and 6.

FIG. 3.

FIG. 3.

Evidence for the overaccumulation of gypsy rasiRNAs antisense strands. Low-molecular-weight enriched RNA was extracted from whole ovaries of the wRev(R) and MG1 restrictive strains (see Materials and Methods for a description of the strains). Three pairs of duplicate Northern blots are shown. They were hybridized with either strand (s, sense; as, antisense) of three RNA probes that are schematized in Fig. 1C. Proper calibration of the pairs of RNA probes was achieved a posteriori by adjusting the Hi display levels of the ImageQuant files (see Materials and Methods) to obtain the same signal intensity on both dsRNA duplicate dot blots that were cohybridized with the Northern blots (not shown). The amount and integrity of the low-molecular-weight RNA loaded in each lane were checked by reprobing the unstripped membranes with the end-labeled oligonucleotide complementary to the abundant mir-13b microRNA (lower panels). The position of the mir-13b 23-nt major band is indicated by black squares. The amounts of gypsy rasiRNAs were quantified with ImageQuant software (Molecular Dynamics) and standardized using those of mir-13 as loading controls and those of the dsRNA dots as probe controls. The amounts of the antisense strands were always three- to fivefold in excess over those of the sense strands, whatever the strain and the fragment used as a probe.

FIG. 4.

FIG. 4.

_flam_-dependent downregulation of transcripts containing the 180-nt sense “_pol_” target. The two reporter constructs described in Fig. 1D, pESpol+ and pESpol−, were introduced by transgenesis into the permissive wOR(P) strain. Several transgenic inserts were then genetically combined with the flamenco genotypes indicated at the top. Their expression was monitored by lacZ staining of whole ovaries. Whole-mount in situ hybridization of ovaries was also performed with a lacZ RNA probe leading to similar observations (not shown). Significant repression was only observed in the MG1(R)/wRev(R) restrictive background and for the four ESpol+ transgenes, whose target is transcribed in the sense orientation (see panel C). Expression of the three ESpol− transgenes did not show any reduction in this restrictive genotype (see panel F). Only the results obtained with two transgenic inserts (F23 and M40) are shown here as examples.

FIG. 5.

FIG. 5.

Effect of three RNA silencing genes on the _flam_-dependent repression of gypsy. Whole-mount in situ hybridization was performed with a digoxigenin-labeled antisense gypsy probe (coordinates 1425 to 199) on either larval female gonads (A and C1) or adult ovaries (B and C2). Larval gonads were oriented so that the anterior is to the top. In panel C1, the magnification is twice the magnification in panel A. Only the ovarioles that contain a stage 10 egg chamber were dissected from the ovaries. Except for a _gypsy_-containing permissive control strain (C), all genotypes were homozygous for the same _gypsy_-containing restrictive X chromosome. Mutant genotypes (m/m = even numbers) were sorted from the corresponding control heterozygous sisters (m/+ = odd numbers) using the dominant phenotypes of the Actin5C-GFP and Cy markers present on the balancer chromosome. The dcr-2 L811fsX (A2 and B2), piwi 1 (A4), and piwi 3 (A6) alleles were tested as homozygous, whereas transheterozygous ovaries, aub QC42 /aub HN2 (A8 and B4) were obtained. Significant derepression of the endogenous gypsy proviruses was only observed in the piwi 1 and piwi 3 homozygous larval female gonads. The reason why gonads were more labeled in A7 and A8 than in A1, A2, A3, and A5 is because, in the aub experiment, the substrate was incubated longer than in the other series of experiments. An additional positive control for a high sensitivity of the digoxigenin detection in this experiment is provided by the strong staining of the gonad-associated fat body (A8, arrowheads), where gypsy is moderately expressed. All adult ovaries exhibited the same restrictive phenotype, consisting in a weak signal restricted to the centripetal and nurse-cell-associated follicle cells.

FIG. 6.

FIG. 6.

piwi and aub are not required for hairpin-induced RNAi in the eye. Shown are typical examples of the level of white repression by the GMR-wIR transgene in the indicated mutant backgrounds. Targeting the white gene by RNAi resulted in a very penetrant yellowish eye color. On the grayscale picture, this repression appears as a much lighter gray (see panels A, B, and D) than the dark gray corresponding to the reproducible reddish eye color seen in the _dcr-2_-null background (see panel C).

FIG. 7.

FIG. 7.

flam is not required for hairpin-induced RNAi in the somatic ovarian tissues. Histochemical staining of the β-galactosidase activity in follicle cells containing either of the ES2 and ES5 transgenic reporters (see Fig. 1D for a description of the constructs). Expression of the lacZ transgene in the follicular epithelium is detected by a dark-blue staining of the X-Gal substrate that appears in black on the grayscale pictures. The presence in the ES5 transgenes of a 59-nt gypsy fragment is known to make them sensitive to the restrictive flam activity (compare panel C with panel G). The UASt_-IRlacZ_ hairpin was expressed in the somatic ovarian tissues by virtue of the activation of its somatic UASt promoter by the ubiquitous Actin5C-Gal4 driver. This resulted in a total RNAi-mediated lacZ repression not only in the presence (B) but also in the absence (F and H) of any flam activity.

Similar articles

Cited by

References

    1. Alefelder, S., B. K. Patel, and F. Eckstein. 1998. Incorporation of terminal phosphorothioates into oligonucleotides. Nucleic Acids Res. 26:4983-4988. - PMC - PubMed
    1. Ambros, V. 2004. The functions of animal microRNAs. Nature 431:350-355. - PubMed
    1. Aravin, A., D. Gaidatzis, S. Pfeffer, M. Lagos-Quintana, P. Landgraf, N. Iovino, P. Morris, M. J. Brownstein, S. Kuramochi-Miyagawa, T. Nakano, M. Chien, J. J. Russo, J. Ju, R. Sheridan, C. Sander, M. Zavolan, and T. Tuschl. 2006. A novel class of small RNAs bind to MILI protein in mouse testes. Nature 442:203-207. - PubMed
    1. Aravin, A., and T. Tuschl. 2005. Identification and characterization of small RNAs involved in RNA silencing. FEBS Lett. 579:5830. - PubMed
    1. Aravin, A. A., M. S. Klenov, V. V. Vagin, F. Bantignies, G. Cavalli, and V. A. Gvozdev. 2004. Dissection of a natural RNA silencing process in the Drosophila melanogaster germ line. Mol. Cell. Biol. 24:6742-6750. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources