Antisense transcription of retro transposable elements in Drosophila: The origin of endogenous small interfering RNA precursors

Unrestricted movement of mobile genetic elements could cause pre-mature lethality in Drosophila melanogaster. Specifically, retro transposons can disrupt genomic integrity through insertions, deletions and chromosomal rearrangements. Therefore, eukaryotes have developed defense mechanisms to silence these elements. In Drosophila, endogenous small interfering (endo-siRNAs) repress retro transposon mobility in somatic cells. The generation of endo-siRNAs requires Dicer-2 processing of double-stranded RNA precursors, yet the origins of this precursor are unknown. Here we show that retro transposons in Dmel-2 cells produce sense and antisense transcripts and identify bonafide transcription start sites for these RNAs. We determine that retro transposon antisense transcripts are less polyadenylated than sense transcripts. RNA-seq and small RNA-seq upon Dicer-2 depletion showed global decrease in endo-siRNAs mapping to retro transposons and increased expression of both S and AS retro transposon transcripts. These data support a model in which double-stranded RNA precursors are derived from convergent transcription and retained in the nucleus. Dicer-2 processes these precursors into endo-siRNAs that silence both sense and antisense retro transposon transcripts. Reduction of sense retro transposon transcripts potentially lowers element specific protein levels required for movement. This mechanism preserves genomic integrity and is especially important for Drosophila fitness because mobile genetic elements are highly active. Overall design: This submission includes 12 raw data files. Two samples (LacZ control and Dcr-2 RNAi-depleted) are represented by three technical triplicate raw data files. RNA-seq and small RNA-seq data are provided for each sample.

移动遗传元件的不受控移动可导致黑腹果蝇（Drosophila melanogaster）出现过早致死现象。具体而言，逆转录转座子（retrotransposons）可通过插入、缺失及染色体重排破坏基因组完整性。因此，真核生物演化出了沉默这类元件的防御机制。在果蝇中，内源性小干扰RNA（endo-siRNAs）可在体细胞中抑制逆转录转座子的移动。内源性小干扰RNA的生成需要Dicer-2对双链RNA（double-stranded RNA）前体进行加工，但这类前体的来源尚不明确。本研究证实，Dmel-2细胞（Dmel-2 cells）中的逆转录转座子可产生正义链与反义链转录本，并鉴定了这些RNA的真实转录起始位点。我们发现，逆转录转座子的反义转录本相较于正义转录本，其多聚腺苷酸化程度更低。对Dicer-2进行敲减后的RNA测序（RNA-seq）与小RNA测序（small RNA-seq）结果显示，靶向逆转录转座子的内源性小干扰RNA整体水平出现下降，同时逆转录转座子的正义与反义转录本的表达量均有所升高。上述数据支持了如下模型：双链RNA前体源自会聚转录，并滞留于细胞核内。Dicer-2将这些前体加工为内源性小干扰RNA，从而沉默正义与反义链的逆转录转座子转录本。减少正义链逆转录转座子的转录本，可潜在降低移动所需的元件特异性蛋白水平。这一机制可维持基因组完整性，对于果蝇的生存适应性尤为关键，因为移动遗传元件的活性极高。总体实验设计：本次提交包含12份原始数据文件。两个样本（LacZ对照与Dcr-2 RNA干扰敲减组）各对应三份技术重复原始数据文件。每个样本均提供了RNA测序与小RNA测序数据。