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De novo piRNA cluster formation in the Drosophila germ line triggered by transgenes containing a transcribed transposon fragment.

Olovnikov I, Ryazansky S, Shpiz S, Lavrov S, Abramov Y, Vaury C, Jensen S, Kalmykova A - Nucleic Acids Res. (2013)

Bottom Line: How these regions are recognized as a source of piRNAs is still elusive.Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences--not only from the transposon fragment.We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.

ABSTRACT
PIWI-interacting RNAs (piRNAs) provide defence against transposable element (TE) expansion in the germ line of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the Long Interspersed Nuclear Elements (LINE)-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions that normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germ line. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences--not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries, the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs.

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Related in: MedlinePlus

Transgene insertions induce generation of small RNAs from flanking genomic sequences. (A–C) Plots of unique small RNAs density, in a 30-bp window, around transgene insertion sites for genomic plus (black) and minus (grey) strand, in transgenic strains 1.9, 2.1, 3.6 and R-strain wK (genomic positions according to dm3 assembly are indicated). Read numbers were normalized to sequencing depths of libraries (rpm). Position and orientation of transgenes are shown by arrowheads and arrows, respectively. The structures of the genomic regions are diagrammed above plots. Insertion of Tirant present in the genome of the sequenced strain (insertion site indicated in B) was not detected in the wK and transgenic strains. (D) Length distribution of small RNAs mapping to flanking genomic regions. Percentages of reads having 1U bias are indicated for each strand. Primers used in the RT–PCR and ChIP are shown (not to scale).
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gkt310-F4: Transgene insertions induce generation of small RNAs from flanking genomic sequences. (A–C) Plots of unique small RNAs density, in a 30-bp window, around transgene insertion sites for genomic plus (black) and minus (grey) strand, in transgenic strains 1.9, 2.1, 3.6 and R-strain wK (genomic positions according to dm3 assembly are indicated). Read numbers were normalized to sequencing depths of libraries (rpm). Position and orientation of transgenes are shown by arrowheads and arrows, respectively. The structures of the genomic regions are diagrammed above plots. Insertion of Tirant present in the genome of the sequenced strain (insertion site indicated in B) was not detected in the wK and transgenic strains. (D) Length distribution of small RNAs mapping to flanking genomic regions. Percentages of reads having 1U bias are indicated for each strand. Primers used in the RT–PCR and ChIP are shown (not to scale).

Mentions: It was previously shown that transgenes containing a fragment of the I-element repressed the I-element activity (20–22). In these constructs, a 2.3-kb I-element fragment (hereinafter referred to as I-TG) had been cloned into the pW8 transgenesis vector between the hsp70 promoter and a sequence containing the actin5C polyadenylation signal (21). Constructs with the I-fragment in sense or antisense orientation were introduced into the genome of the Drosophila reactive wK strain. We confirmed that at present, all transgenic strains used in this study are characterized by very low-reactivity levels (data not shown). To address the mechanism of the repressive effect of an I-containing transgene, we sequenced small RNAs from ovaries of wK and transgenic strains (Supplementary Figure S1 and Supplementary Table S2). We analysed five strains with an I-sense construct (1.9, 2.1, 2.3, 2.6 and 2.10), four with an I-antisense (3.1, 3.6, 3.9 and 3.10), one strain with a construct containing the I-TG but no promoter, one control strain with a construct missing the I-TG region (strain 62.5.2) and the reactive wK strain (21) (Supplementary Figure S2). For all of the transgenic strains, insertion sites were determined using inverse-PCR (Supplementary Table S1). In strain 3.1, the transgene was inserted into 3R telomere-associated sequences (TAS), which is a potent piRNA cluster; in the other strains, the insertions were located in euchromatic regions not adjacent to piRNA clusters. Insertion of TE Tirant in gene CG32486 present in the genome of the sequenced strain (insertion site indicated in Figure 4B) was not detected in the wK and transgenic strains.


De novo piRNA cluster formation in the Drosophila germ line triggered by transgenes containing a transcribed transposon fragment.

Olovnikov I, Ryazansky S, Shpiz S, Lavrov S, Abramov Y, Vaury C, Jensen S, Kalmykova A - Nucleic Acids Res. (2013)

Transgene insertions induce generation of small RNAs from flanking genomic sequences. (A–C) Plots of unique small RNAs density, in a 30-bp window, around transgene insertion sites for genomic plus (black) and minus (grey) strand, in transgenic strains 1.9, 2.1, 3.6 and R-strain wK (genomic positions according to dm3 assembly are indicated). Read numbers were normalized to sequencing depths of libraries (rpm). Position and orientation of transgenes are shown by arrowheads and arrows, respectively. The structures of the genomic regions are diagrammed above plots. Insertion of Tirant present in the genome of the sequenced strain (insertion site indicated in B) was not detected in the wK and transgenic strains. (D) Length distribution of small RNAs mapping to flanking genomic regions. Percentages of reads having 1U bias are indicated for each strand. Primers used in the RT–PCR and ChIP are shown (not to scale).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675497&req=5

gkt310-F4: Transgene insertions induce generation of small RNAs from flanking genomic sequences. (A–C) Plots of unique small RNAs density, in a 30-bp window, around transgene insertion sites for genomic plus (black) and minus (grey) strand, in transgenic strains 1.9, 2.1, 3.6 and R-strain wK (genomic positions according to dm3 assembly are indicated). Read numbers were normalized to sequencing depths of libraries (rpm). Position and orientation of transgenes are shown by arrowheads and arrows, respectively. The structures of the genomic regions are diagrammed above plots. Insertion of Tirant present in the genome of the sequenced strain (insertion site indicated in B) was not detected in the wK and transgenic strains. (D) Length distribution of small RNAs mapping to flanking genomic regions. Percentages of reads having 1U bias are indicated for each strand. Primers used in the RT–PCR and ChIP are shown (not to scale).
Mentions: It was previously shown that transgenes containing a fragment of the I-element repressed the I-element activity (20–22). In these constructs, a 2.3-kb I-element fragment (hereinafter referred to as I-TG) had been cloned into the pW8 transgenesis vector between the hsp70 promoter and a sequence containing the actin5C polyadenylation signal (21). Constructs with the I-fragment in sense or antisense orientation were introduced into the genome of the Drosophila reactive wK strain. We confirmed that at present, all transgenic strains used in this study are characterized by very low-reactivity levels (data not shown). To address the mechanism of the repressive effect of an I-containing transgene, we sequenced small RNAs from ovaries of wK and transgenic strains (Supplementary Figure S1 and Supplementary Table S2). We analysed five strains with an I-sense construct (1.9, 2.1, 2.3, 2.6 and 2.10), four with an I-antisense (3.1, 3.6, 3.9 and 3.10), one strain with a construct containing the I-TG but no promoter, one control strain with a construct missing the I-TG region (strain 62.5.2) and the reactive wK strain (21) (Supplementary Figure S2). For all of the transgenic strains, insertion sites were determined using inverse-PCR (Supplementary Table S1). In strain 3.1, the transgene was inserted into 3R telomere-associated sequences (TAS), which is a potent piRNA cluster; in the other strains, the insertions were located in euchromatic regions not adjacent to piRNA clusters. Insertion of TE Tirant in gene CG32486 present in the genome of the sequenced strain (insertion site indicated in Figure 4B) was not detected in the wK and transgenic strains.

Bottom Line: How these regions are recognized as a source of piRNAs is still elusive.Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences--not only from the transposon fragment.We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia.

ABSTRACT
PIWI-interacting RNAs (piRNAs) provide defence against transposable element (TE) expansion in the germ line of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the Long Interspersed Nuclear Elements (LINE)-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions that normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germ line. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences--not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries, the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs.

Show MeSH
Related in: MedlinePlus