Tau aggregates are RNA-protein assemblies that mis-localize multiple nuclear speckle components. Tau aggregates are RNA-protein assemblies that mis-localize multiple nuclear speckle components

We report the isolation and sequencing of tau aggregates from [1] HEK293 cells expressing Tau-RD-P301S-CFP/YFP that have been seeded with preformed fibrils from the brain of P301S mice (B6-Tg(Thy1-MAPT*P301S)2541; referred to as Tg2541 mice). Tau aggregates were isolated by differential centrifugation followed by fluorescence automated particle sorting using a BD FACSaraia. We found that these tau aggregates were enriched for particular small non-coding RNAs, including snoRNAs and snRNAs. [2] the following mice: FvBB6F1-Tg(Camk2a-tTa),(tetO-MAPT*wt)21221 (referred to as rTg21221 or WT tau mice in the paper) and FvBB6F1-Tg(Camk2a-tTA)1Mmay, (tet)-tdTomato-Syp/EGFP)1.1Luo/J,(tetO-MAPT*P301L)4510 (referred to as rTg4510 or P301L mice in the paper). Briefly, tau aggregates were isolated by 1% sarkosyl extraction (to enrich for insoluble proteins) followed by immunoprecipitation of tau using the tau-12 antibody (see Methods section of associated paper for further details). We found that these tau aggregates were enriched for particular small non-coding RNAs, including snRNAs and some snoRNAs. [3] Sequencing of HEK293 tau biosensor cells with and without tau aggregates reveals evidence of significant splicing alterations. Specifically we observed an increase in intron retention events in cells that contain tau aggregates relative to cells without tau aggregates. Overall design: Examination of the RNA composition of tau aggregates in HEK293 cells [GSM4477465-GSM4477468] and from the brains of P301L mice (rTg4510) [GSM4484210-GSM4484221]. Examination of total RNA in HEK293 tau biosensor cells with and without tau aggregates [GSM5227473-GSM5227478].

本研究报道了来自[1]表达Tau-RD-P301S-CFP/YFP的HEK293细胞（HEK293 cells）中tau聚集体（tau aggregates）的分离与测序：该细胞系经来自P301S小鼠（P301S mice，品系为B6-Tg(Thy1-MAPT*P301S)2541，下称Tg2541小鼠）大脑中的预形成纤维（preformed fibrils）诱导接种。本研究通过差速离心结合BD FACSaria流式细胞分选仪的荧光自动颗粒分选步骤，完成了此类tau聚集体的分离。经分析发现，该类tau聚集体富集特定小型非编码RNA（small non-coding RNAs），包括核仁小RNA（small nucleolar RNAs, snoRNAs）与核内小RNA（small nuclear RNAs, snRNAs）。
[2] 本研究涉及以下品系小鼠：FvBB6F1-Tg(Camk2a-tTa)、(tetO-MAPT*wt)21221（论文中记作rTg21221或野生型tau小鼠），以及FvBB6F1-Tg(Camk2a-tTA)1Mmay、(tet)-tdTomato-Syp/EGFP)1.1Luo/J、(tetO-MAPT*P301L)4510（论文中记作rTg4510或P301L小鼠）。简言之，该类tau聚集体通过1%十二烷基肌氨酸钠（sarkosyl）提取以富集不溶性蛋白，随后使用tau-12抗体（tau-12 antibody）对tau蛋白进行免疫沉淀分离（详细操作步骤参见相关论文的方法部分）。分析结果显示，此类tau聚集体同样富集特定小型非编码RNA，包括核内小RNA（snRNAs）及部分核仁小RNA（snoRNAs）。
[3] 对含与不含tau聚集体的HEK293 tau生物传感器细胞（HEK293 tau biosensor cells）进行测序，结果显示存在显著剪接改变（splicing alterations）证据：具体而言，与不含tau聚集体的细胞相比，含tau聚集体的细胞中内含子保留（intron retention）事件显著增多。
整体实验设计如下：① 分析HEK293细胞[GSM4477465-GSM4477468]与P301L小鼠（rTg4510）大脑[GSM4484210-GSM4484221]中tau聚集体的RNA组成；② 分析含与不含tau聚集体的HEK293 tau生物传感器细胞的总RNA（total RNA）[GSM5227473-GSM5227478]。