Characterization of the RNA present in L1 ORF1p macromolecular complexes from Mael-/- mouse testes

piRNA-deficient Maelstrom (Mael) null mice are characterized by a strong upregulation of LINE-1 (L1) retrotransposon in meiotic spermatocytes. This defect turns out in the accumulation of L1 RNA and ORF1p in their cytoplasm and the formation of prominent ribonucleoprotein aggregates. We used 3-months-old Mael-/- male mice to characterize the RNA present in those ORF1p aggregates. To favor the isolation of complexed versus free ORF1p protein, we first fractionated Mael-/- testis extracts (that we refer to as TOTAL) by sucrose gradient ultracentrifugation, in the presence of EDTA. We then pooled the sucrose fractions where ORF1p macromolecular complexes sediment (fractions 5-8) and used this pool as the INPUT for an anti-ORF1p co-immunoprecipitation (IP) followed by RNA-seq. Enrichment of RNAs in anti-ORF1p co-immunoprecipitation samples (IP) was calculated over the background controls (Beads Only - BO), over the pools of sucrose fractions 5-8 (INPUT), and over the testis extracts originally loaded on sucrose gradients (TOTAL). Three biological replicates are reported.piRNA缺陷型Maelstrom（Mael）敲除小鼠的特征为减数分裂精母细胞中LINE-1（L1）逆转座子显著上调。该缺陷表现为L1 RNA与开放阅读框1蛋白（ORF1p）在胞浆中积累，并形成显著的核糖核蛋白聚集体。本研究使用3月龄的Mael基因敲除雄性小鼠，对上述ORF1p聚集体中存在的RNA进行表征。为优先分离结合态而非游离态的ORF1p蛋白，我们首先在乙二胺四乙酸（EDTA）存在的条件下，通过蔗糖梯度超速离心对Mael-/-小鼠的睾丸提取物（本研究中记为"TOTAL"）进行分级分离。随后收集ORF1p大分子复合物沉降所在的蔗糖梯度组分（组分5-8），将该混合组分作为"INPUT"样本，用于靶向ORF1p的免疫共沉淀（co-immunoprecipitation，IP）实验，后续对免疫沉淀获得的RNA进行RNA测序（RNA-seq）。我们以空白对照（仅磁珠组，Beads Only - BO）、蔗糖梯度组分5-8混合"INPUT"样本、原始加载至蔗糖梯度的睾丸提取物"TOTAL"样本为参照，计算靶向ORF1p免疫共沉淀样本（IP）中RNA的富集水平。本研究共设置3次生物学重复。