Parallel high-throughput RNA-sequencing suggests little overlap of differential gene expression between knockout of TDP-43 and its over-expression in central nervous system in Drosophila

The human Tar-DNA-binding protein TDP-43 is closely associated with ALS and other neurodegenerative disorders. TDP-43 contains two highly conserved RNA-binding motifs and possesses a variety of documented roles in RNA metabolism, including pre-RNA splicing and repression of transcription. We sought to measure the effect that knockout and over-expression of the fly orthologue of this protein, Tar-DNA-binding protein homolog (TBPH), has on the transcriptome of the central nervous system (CNS) of Drosophila melanogaster. To this end, we used massively parallel sequencing methods (RNA-seq) to transcriptionally profile the CNS in loss-of-function mutants and gain-of-function over-expression genotypes. We found that loss of TBPH resulted in widespread gene activation, much of which could be reversed by rescue of TBPH expression, suggesting that repression is one of the major roles of TBPH. Conversely, we found that over-expression of TBPH resulted largely in decreased gene expression. However, there was little overlap in the genes which were affected in these two genotypes, suggesting that the bulk of genes affected by TBPH loss-of-function and over-expression are different. We provide a comprehensive look at enriched gene ontologies in both cases, suggesting that TDP-43 plays a role in regulating basic processes in neurons. We also describe a number of genes whose splicing is likely to be altered in the absence of TDP-43. Overall design: In this study we compare the effects of knockout of the TDP-43 ortholog (TBPH) in the fly nervous system with the effects of its overexpression. 2 treatment groups are presented. In the first, TDP-43 knockout (G2) is compared to control (A1) and rescue (G2; TDP-43-GAL4>UAS-TDP-43). In the second comparison, the motor neuron driver D42-GAL4 is used to overexpress TDP-43 in motor neurons, using LacZ as a control for overexpression of a foreign protein.

人类Tar DNA结合蛋白TDP-43（Tar-DNA-binding protein TDP-43）与肌萎缩侧索硬化症（ALS）及其他神经退行性疾病密切相关。TDP-43包含两个高度保守的RNA结合基序，在RNA代谢过程中具备多种已被证实的功能，包括前体RNA剪接与转录抑制。本研究旨在探究该蛋白的果蝇同源物——Tar DNA结合蛋白同源物（TBPH，Tar-DNA-binding protein homolog）的敲除与过表达对黑腹果蝇（Drosophila melanogaster）中枢神经系统（CNS）转录组的影响。为此，我们采用大规模并行测序技术（RNA测序，RNA-seq），对功能丧失突变体与功能获得性过表达基因型的中枢神经系统进行转录组谱分析。研究结果显示，TBPH缺失会导致广泛的基因激活，其中多数激活现象可通过TBPH表达拯救得以逆转，这表明基因抑制是TBPH的主要功能之一。与之相反，TBPH过表达主要会引发基因表达水平下调。然而，这两种基因型中受影响的基因重叠度极低，提示TBPH功能丧失与过表达所影响的绝大多数基因存在差异。本研究对两种情况下的富集基因本体（gene ontologies）进行了全面分析，结果表明TDP-43在调控神经元基础生理过程中发挥作用。此外，我们还鉴定出了一系列在TDP-43缺失状态下可能发生剪接异常的基因。实验整体设计：本研究对比了果蝇TDP-43同源基因TBPH敲除及其过表达对神经系统的影响，共设置2个处理组。第一组中，我们将TDP-43敲除型（G2）与对照组（A1）以及TBPH表达拯救型（G2; TDP-43-GAL4>UAS-TDP-43）进行对比。第二组中，我们利用运动神经元驱动因子D42-GAL4在运动神经元中过表达TDP-43，并以LacZ作为外源蛋白过表达的对照。