Telomere elongation is under the control of the RNAi-based mechanism in the Drosophila germline - PubMed (original) (raw)

Telomere elongation is under the control of the RNAi-based mechanism in the Drosophila germline

Mikhail Savitsky et al. Genes Dev. 2006.

Abstract

Telomeres in Drosophila are maintained by transposition of specialized telomeric retroelements HeT-A, TAHRE, and TART instead of the short DNA repeats generated by telomerase in other eukaryotes. Here we implicate the RNA interference machinery in the control of Drosophila telomere length in ovaries. The abundance of telomeric retroelement transcripts is up-regulated owing to mutations in the spn-E and aub genes, encoding a putative RNA helicase and protein of the Argonaute family, respectively, which are related to the RNA interference (RNAi) machinery. These mutations cause an increase in the frequency of telomeric element retrotransposition to a broken chromosome end. spn-E mutations eliminate HeT-A and TART short RNAs in ovaries, suggesting an RNAi-based mechanism in the control of telomere maintenance in the Drosophila germline. Enhanced frequency of TART, but not HeT-A, attachments in individuals carrying one dose of mutant spn-E or aub alleles suggests that TART is a primary target of the RNAi machinery. At the same time, we detected enhanced HeT-A attachments to broken chromosome ends in oocytes from homozygous spn-E mutants. Double-stranded RNA (dsRNA)-mediated control of telomeric retroelement transposition may occur at premeiotic stages, resulting in the maintenance of appropriate telomere length in gamete precursors.

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Figures

Figure 1.

Figure 1.

Effect of spn-E and aub mutations on the abundance of HeT-A and TART transcripts in ovaries observed by in situ RNA hybridization. (A) TART riboprobes detecting sense (s, upper panels) and antisense (as, lower panels) transcripts. TART sense transcripts are detected in the cytoplasm of nurse cells at the late stage of oogenesis in spn-E1/spn-Ehls3987 and aubQC42/aub_Δ_P-3a ovaries. Identical staining was observed in aubQC42/aubHN, aubQC42/aubN11, and aubQC42/aubsting-1 ovaries (data not shown). TART antisense transcript abundance is not affected by spn-E and aub mutations. (B) Detection of HeT-A sense transcripts in aub ovaries. HeT-A transcripts are detected in the cytoplasm of nurse cells and in the growing oocyte (arrows) in aubQC42/aub_Δ_P-3a ovaries. The two panels on the right show the mosaic character of HeT-A expression detected only in separate ovarioles. Identical staining was observed in aubQC42/aubHN and aubQC42/aubN11 (data not shown). The _aubQC42/aubsting-1 trans_-heterozygotes show no effect on HeT-A transcript abundance (data not shown). (C) Detection of sense HeT-A transcripts in eye-antennal discs of spn-E larvae. No difference in HeT-A expression in the cells of the morphogenetic furrow of eye-antennal discs of spn-E1/+ and spn-E1/spn-E1 larvae is observed. Genotypes are indicated at the top of panels.

Figure 2.

Figure 2.

Molecular structure of the HeT-A/TART attachments to the broken chromosome end. (A) Yellow (left)-to-black (right) change in pigmentation of aristae (indicating by arrows) is a result of addition of HeT-A or TART retroelements to the broken X chromosome truncated upstream of the yellow gene. (B) Schematic representation of HeT-A/TART attachments to the broken yellow gene. The yellow region is shown as a yellow box. The horizontal red and green arrows show HeT-A/TART attachments, respectively. Vertical arrows point to EcoRI restriction sites. The solid horizontal line indicates a yellow probe used for Southern analysis. The junctions between yellow and new DNA attachments were amplified using primers schematically indicated as small horizontal arrows. (C) Southern blot analysis of DNAs prepared from the progeny of individual yTD/y ac; spn-E1/MKRS flies with yellow aristae (without attachments, lane 10) and black aristae (with TART or HeT-A attachments, lanes 1-6 and 7-9, respectively). DNA was digested by EcoRI enzyme. The filter was hybridized with the yellow probe indicated in B. The 4-kb fragment in lane 10 reflects the distance from the EcoRI site in yellow to the truncated end of the broken chromosome. The 5-15-kb bands in lanes 1-6 correspond to the different size attachments of TART elements. The 5.0-kb bands in lanes 7-10 correspond to attachments of HeT-A elements carrying a conservative EcoRI site in the 3′ region. (D) Southern analysis of DNA isolated from F2 progeny of four females with black aristae (TART attachments), the descendants of an individual yTD/y ac; aubQC42/CyO fly with yellow aristae. Hybridized fragments are identical, confirming TART addition at the earlier premeiotic stages of oogenesis. DNA size markers are indicated in kilobases.

Figure 3.

Figure 3.

Suppression effect of spn-E on TART and HeT-A expression. RT-PCR analysis of TART (A) and HeT-A (B) transcript amounts in ovaries of yTD/y ac; spn-E1/MKRS (spn-E/+) and yTD/y ac; spn-E1/spn-Ehls3987 (spn-E/spn-E) flies. Histograms display the quantification of five experiments. Bars represent the ratios of TART or HeT-A to rp49 transcript abundance in yTD/y ac; spn-E1/MKRS (spn-E/+) and yTD/y ac; spn-E1/spn-Ehls3987 (spn-E/spn-E) ovaries related to this ratio in yTD/y ac; TM6/MKRS (+/+). Dosage effect of spn-E on TART expression is demonstrated (A), while drastic accumulation of HeT-A transcripts is observed only in ovaries of spn-E/spn-E flies (B).

Figure 4.

Figure 4.

Effect of spn-E mutation on the presence of the short HeT-A and TART RNAs in ovaries. Northern analysis of RNA, isolated from ovaries of Gaiano (+/+), spn-E1/+ (+/-), and spn-E1/spn-Ehls3987 (-/-) flies. (A) Hybridization with the TART sense riboprobe reveals short TART RNAs of 27-29 nt in size in Gaiano, a faint signal in heterozygous ovaries, and no signal in ovaries of heteroallelic spn-E flies. (B) Hybridization with the HeT-A sense riboprobe. Short HeT-A RNAs are absent in heteroallelic spn-E1/spn-Ehls3987 (-/-) flies. The lower panels represent hybridization with an oligonucleotide complementary to the mir-13b1 microRNA. 33P-labeled RNA oligonucleotides were used as size markers.

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