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Argonaute protein PIWI controls mobilization of retrotransposons in the Drosophila male germline.

Kalmykova AI, Klenov MS, Gvozdev VA - Nucleic Acids Res. (2005)

Bottom Line: The piwi gene encoding protein of the Drosophila Argonaute family was shown to be required for the germ stem cells maintenance.Thus, piwi is involved in the silencing of retrotransposons in the precursors of male gametes.Our results provide the first evidence that protein of the Argonaute family prevents retrotranspositions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Genetics RAS Kurchatov square 2, 123 182 Moscow, Russia.

ABSTRACT
Proteins of the Argonaute family have been identified as key components of RNA interference (RNAi) pathway. RNAi-related mechanisms are implicated in the regulation of gene expression and repression of transposable elements in eukaryotes. The piwi gene encoding protein of the Drosophila Argonaute family was shown to be required for the germ stem cells maintenance. Here, we show that piwi is involved in silencing of LTR retrotransposons in testes. piwi mutations led to derepression of endogenous retrotransposon copia as well as to upregulation of the reporter gene driven by copia LTR. piwi mutation causes accumulation of retrotransposon mdg1 transcripts at the apical tip of testes, including germinal proliferative center where PIWI protein was shown to be expressed. We applied inverse PCR approach to detect the newly arisen insertions of the mdg1 retrotransposon in the progeny of individual piwi mutant males. Owing to piwi mutation a high rate of mdg1 transpositions was revealed. Thus, piwi is involved in the silencing of retrotransposons in the precursors of male gametes. Our results provide the first evidence that protein of the Argonaute family prevents retrotranspositions. It is supposed that the disturbance of RNA silencing system in germinal cells might cause transposition burst.

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Overexpression of the copiaLTR-lacZ construct in piwi2 and spn-E1 testes. (A) The copiaLTR-lacZ construct comprises full-size copia LTR fused to the lacZ reporter. (B) X-gal staining of testes from piwi2 males carrying the copiaLTR-lacZ construct on the X-chromosome. (C) X-gal staining of testes from spn-E1 males carrying the copiaLTR-lacZ construct on the X (upper panels) and second chromosomes (lower panels). A level of lacZ expression is greatly increased in the homozygous piwi/piwi and spn-E/spn-E testes as compared with heterozygous piwi/+ and spn-E/+. Arrows indicate the apical tips of testes, where β-galactosidase activity is not detected.
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fig2: Overexpression of the copiaLTR-lacZ construct in piwi2 and spn-E1 testes. (A) The copiaLTR-lacZ construct comprises full-size copia LTR fused to the lacZ reporter. (B) X-gal staining of testes from piwi2 males carrying the copiaLTR-lacZ construct on the X-chromosome. (C) X-gal staining of testes from spn-E1 males carrying the copiaLTR-lacZ construct on the X (upper panels) and second chromosomes (lower panels). A level of lacZ expression is greatly increased in the homozygous piwi/piwi and spn-E/spn-E testes as compared with heterozygous piwi/+ and spn-E/+. Arrows indicate the apical tips of testes, where β-galactosidase activity is not detected.

Mentions: To study whether copia LTR is a target of silencing mediated by piwi, we used a construct containing full-size copia LTR fused to lacZ (copiaLTR-lacZ). copia LTR comprises the known upstream regulatory region, including 137 bp of the transcribed fragment (54) (Figure 2A). We introduced theX-chromosome containing copiaLTR-lacZ into the stock carrying piwi2 mutation. The level of reporter gene expression is significantly increased in piwi2/piwi2 testes. No staining at the apical tip of testes is revealed (Figure 2B, arrows). lacZ expression is detected in early spermatocytes as well as in the basal parts of piwi2 homozygous testes where the accumulation of endogenous copia transcripts was observed (Figures 1B and 2B). The difference in the expression patterns of endogenous copia and copiaLTR-lacZ might be related to the absence of some copia regulatory regions in the transgene as compared with full-size endogenous copies.


Argonaute protein PIWI controls mobilization of retrotransposons in the Drosophila male germline.

Kalmykova AI, Klenov MS, Gvozdev VA - Nucleic Acids Res. (2005)

Overexpression of the copiaLTR-lacZ construct in piwi2 and spn-E1 testes. (A) The copiaLTR-lacZ construct comprises full-size copia LTR fused to the lacZ reporter. (B) X-gal staining of testes from piwi2 males carrying the copiaLTR-lacZ construct on the X-chromosome. (C) X-gal staining of testes from spn-E1 males carrying the copiaLTR-lacZ construct on the X (upper panels) and second chromosomes (lower panels). A level of lacZ expression is greatly increased in the homozygous piwi/piwi and spn-E/spn-E testes as compared with heterozygous piwi/+ and spn-E/+. Arrows indicate the apical tips of testes, where β-galactosidase activity is not detected.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1074743&req=5

fig2: Overexpression of the copiaLTR-lacZ construct in piwi2 and spn-E1 testes. (A) The copiaLTR-lacZ construct comprises full-size copia LTR fused to the lacZ reporter. (B) X-gal staining of testes from piwi2 males carrying the copiaLTR-lacZ construct on the X-chromosome. (C) X-gal staining of testes from spn-E1 males carrying the copiaLTR-lacZ construct on the X (upper panels) and second chromosomes (lower panels). A level of lacZ expression is greatly increased in the homozygous piwi/piwi and spn-E/spn-E testes as compared with heterozygous piwi/+ and spn-E/+. Arrows indicate the apical tips of testes, where β-galactosidase activity is not detected.
Mentions: To study whether copia LTR is a target of silencing mediated by piwi, we used a construct containing full-size copia LTR fused to lacZ (copiaLTR-lacZ). copia LTR comprises the known upstream regulatory region, including 137 bp of the transcribed fragment (54) (Figure 2A). We introduced theX-chromosome containing copiaLTR-lacZ into the stock carrying piwi2 mutation. The level of reporter gene expression is significantly increased in piwi2/piwi2 testes. No staining at the apical tip of testes is revealed (Figure 2B, arrows). lacZ expression is detected in early spermatocytes as well as in the basal parts of piwi2 homozygous testes where the accumulation of endogenous copia transcripts was observed (Figures 1B and 2B). The difference in the expression patterns of endogenous copia and copiaLTR-lacZ might be related to the absence of some copia regulatory regions in the transgene as compared with full-size endogenous copies.

Bottom Line: The piwi gene encoding protein of the Drosophila Argonaute family was shown to be required for the germ stem cells maintenance.Thus, piwi is involved in the silencing of retrotransposons in the precursors of male gametes.Our results provide the first evidence that protein of the Argonaute family prevents retrotranspositions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Genetics RAS Kurchatov square 2, 123 182 Moscow, Russia.

ABSTRACT
Proteins of the Argonaute family have been identified as key components of RNA interference (RNAi) pathway. RNAi-related mechanisms are implicated in the regulation of gene expression and repression of transposable elements in eukaryotes. The piwi gene encoding protein of the Drosophila Argonaute family was shown to be required for the germ stem cells maintenance. Here, we show that piwi is involved in silencing of LTR retrotransposons in testes. piwi mutations led to derepression of endogenous retrotransposon copia as well as to upregulation of the reporter gene driven by copia LTR. piwi mutation causes accumulation of retrotransposon mdg1 transcripts at the apical tip of testes, including germinal proliferative center where PIWI protein was shown to be expressed. We applied inverse PCR approach to detect the newly arisen insertions of the mdg1 retrotransposon in the progeny of individual piwi mutant males. Owing to piwi mutation a high rate of mdg1 transpositions was revealed. Thus, piwi is involved in the silencing of retrotransposons in the precursors of male gametes. Our results provide the first evidence that protein of the Argonaute family prevents retrotranspositions. It is supposed that the disturbance of RNA silencing system in germinal cells might cause transposition burst.

Show MeSH
Related in: MedlinePlus