TDP-43 loss induces cryptic polyadenylation in ALS/FTD [shsy5y-chx-tdpkd-vs-ctl]

Nuclear depletion and cytoplasmic aggregation of the RNA-binding protein TDP-43 is the hallmark of ALS, occurring in over 97% of cases. A key consequence of TDP-43 nuclear loss is the de-repression of cryptic exons. Whilst TDP-43 regulated cryptic splicing is increasingly well catalogued, cryptic alternative polyadenylation (APA) events, which define the 3’ end of last exons, have been largely overlooked, especially when not associated with novel upstream splice junctions. We developed a bioinformatic pipeline to reliably identify distinct APA event types: alternative last exons (ALE), 3’UTR extensions (3’Ext) and ‘ composite ’ intronic polyadenylation (IPA) events. We identified novel neuronal cryptic APA sites induced by TDP-43 loss of function by systematically applying our pipeline to a compendium of publicly available and in house datasets. We find that TDP-43 binding sites and target motifs are enriched at these cryptic events and that TDP-43 can have both repressive and enhancing action on APA. Importantly, all categories of cryptic APA can also be identified in ALS and FTD post mortem brain regions with TDP-43 proteinopathy underlining their potential disease relevance. RNA-seq and Ribo-seq analyses indicate that distinct cryptic APA categories have different downstream effects on transcript and translation. Intriguingly, cryptic 3’Exts occur in multiple transcription factors, such as ELK1 , SIX3, and TLX1, and lead to an increase in RNA stability. 3’Exts are localised to the cytoplasm and neurites and translated leading to an increase in wild-type protein levels. In summary, we demonstrate that TDP-43 nuclear depletion induces a novel category of cryptic RNA processing events and we expand the palette of TDP-43 loss consequences by showing this can also lead to an increase in normal protein translation. short-read bulk RNA-sequencing of the SH-SY5Y neuroblastoma cell line with and without TDP-43 knockdown (4 controls, 4 knockdowns)

RNA结合蛋白TDP-43的核耗竭与细胞质聚集是肌萎缩侧索硬化症（ALS）的标志性病理特征，在超过97%的ALS病例中均可观察到该现象。TDP-43的核丢失所引发的关键后果之一，是隐蔽外显子的去抑制。尽管TDP-43调控的隐蔽剪接事件已得到越来越充分的编目，但定义最后外显子3'端的隐蔽可变多聚腺苷酸化（alternative polyadenylation, APA）事件在很大程度上被忽视了，尤其是在不与新的上游剪接位点相关联的情况下。我们开发了一套生物信息学分析流程，可稳定鉴定出不同类型的APA事件：可变最后外显子（alternative last exons, ALE）、3'非翻译区延伸（3'UTR extensions, 3'Ext）以及复合内含子多聚腺苷酸化（composite intronic polyadenylation, IPA）事件。通过将该分析流程系统性应用于公开及自研数据集的合集，我们成功鉴定出了由TDP-43功能丧失所诱导的新型神经元源性隐蔽APA位点。研究发现，TDP-43结合位点与靶基序在这些隐蔽APA事件中显著富集，且TDP-43对APA事件同时具有抑制与增强双重调控作用。值得注意的是，在伴有TDP-43蛋白质病的ALS及额颞叶痴呆（FTD）死后脑区中，同样可检测到各类隐蔽APA事件，这进一步凸显了这类事件与疾病的潜在关联。RNA测序（RNA-seq）与核糖体测序（Ribo-seq）分析结果显示，不同类型的隐蔽APA事件对转录本及翻译过程具有各异的下游调控效应。有趣的是，隐蔽3'UTR延伸事件可发生于ELK1、SIX3及TLX1等多种转录因子基因中，并可提升对应转录本的RNA稳定性。这类3'UTR延伸转录本可定位于细胞质与神经突中并被翻译，进而使野生型蛋白的表达水平上调。综上，本研究证实TDP-43核耗竭可诱导一类新型的隐蔽RNA加工事件，并通过揭示该过程还可促进正常蛋白的翻译，进一步拓展了TDP-43功能丧失所引发的生物学后果范畴。本数据集包含对TDP-43敲低及未敲低的SH-SY5Y神经母细胞瘤细胞系进行的短读长批量RNA测序（共4例对照样本、4例敲低样本）。