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

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.

SUBMITTER: Pelisson A

PROVIDER: S-EPMC1797544 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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

Pélisson Alain A Sarot Emeline E Payen-Groschêne Geneviève G Bucheton Alain A

Journal of virology 20061129 4

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 ...[more]

PMID: 17135323

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Project description:BackgroundThe establishment of facultative heterochromatin by X-chromosome inactivation requires the long non-coding RNA XIST/Xist. However, the molecular mechanism by which the RNA achieves chromosome-wide gene silencing remains unknown. Mouse Xist has been shown to have redundant domains for cis-localization, and requires a series of well-conserved tandem 'A' repeats for silencing. We previously described a human inducible XIST transgene that is capable of cis-localization and suppressing a downstream reporter gene in somatic cells, and have now leveraged these cells to dissect the sequences critical for XIST-dependent gene silencing in humans.ResultsWe demonstrated that expression of the inducible full-length XIST cDNA was able to suppress expression of two nearby reporter genes as well as endogenous genes up to 3 MB from the integration site. An inducible construct containing the repeat A region of XIST alone could silence the flanking reporter genes but not the more distal endogenous genes. Reporter gene silencing could also be accomplished by a synthetic construct consisting of nine copies of a consensus repeat A sequence, consistent with previous studies in mice. Progressively shorter constructs showed a linear relationship between the repeat number and the silencing capacity of the RNA. Constructs containing only two repeat A units were still able to partially silence the reporter genes and could thus be used for site-directed mutagenesis to demonstrate that sequences within the two palindromic cores of the repeat are essential for silencing, and that it is likely the first palindrome sequence folds to form a hairpin, consistent with compensatory mutations observed in eutherian sequences.ConclusionsSilencing of adjacent reporter genes can be effected by as little as 94 bp of XIST, including two 'monomers' of the A repeat. This region includes a pair of essential palindromic sequences that are evolutionarily well-conserved and the first of these is likely to form an intra-repeat hairpin structure. Additional sequences are required for the spread of silencing to endogenous genes on the chromosome.

Dataset Information

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

Publications

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

Similar Datasets

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