TDP-43 regulates β-adducin (Add2) transcript stability
TDP-43 regulates β-adducin (Add2) transcript stability
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DOI：
10.1080/15476286.2014.996081
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发表时间：
2014-10-01
期刊：
影响因子：
4.1
通讯作者：
Muro, Andres F.
中科院分区：
文献类型：
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作者：
Costessi, Luisa;Porro, Fabiola;Muro, Andres F.
TDP-43是一种RNA结合蛋白，参与mRNA代谢的几个步骤，包括转录、剪接和稳定性。它也参与ALS和FTD，以TDP-43核耗竭为特征的神经退行性疾病。我们以前确定TDP-43作为-内收蛋白（Add 2）脑特异性多聚腺苷酸化位点（A4 PAS）的下游元件（DSE）的结合剂，表明其参与前mRNA 3端加工。在这里，通过使用嵌合小基因，我们表明HeLa和HEK 293细胞中的TDP-43消耗导致嵌合和内源性Add 2转录物的下调。尽管已经证实了TDP-43-DSE的体外相互作用，但我们证明了体内效应不是由TDP-43-DSE相互作用介导的。事实上，用不是TDP-43靶标的病毒E-SV 40和L-SV 40 DSE取代Add 2 DSE，在TDP-43下调后仍导致Add 2 mRNA水平降低。此外，我们未能显示TDP-43与关键的多聚腺苷酸化因子（如CstF-64和CPSF 160）之间的相互作用，并且排除了TDP-43参与前mRNA切割和polyA尾长的调节。这些证据允许我们排除预先假设的TDP 43在调节Add 2前mRNA的3端加工中的作用。最后，我们发现TDP-43通过增加Add 2 mRNA的稳定性来调节Add 2基因的表达水平。考虑到大脑中的Add 2参与突触组装、突触可塑性及其稳定性，并且其在小鼠中的遗传失活导致LTP、LTD、学习和运动协调缺陷，我们假设TDP-43耗尽可能导致Add 2功能丧失可能导致ALS和FTD疾病状态。
TDP-43 is an RNA-binding protein involved in several steps of mRNA metabolism including transcription, splicing and stability. It is also involved in ALS and FTD, neurodegenerative diseases characterized by TDP-43 nuclear depletion. We previously identified TDP-43 as a binder of the downstream element (DSE) of the -Adducin (Add2) brain-specific polyadenylation site (A4 PAS), suggesting its involvement in pre-mRNA 3 end processing. Here, by using chimeric minigenes, we showed that TDP-43 depletion in HeLa and HEK293 cells resulted in down-regulation of both the chimeric and endogenous Add2 transcripts. Despite having confirmed TDP-43-DSE in vitro interaction, we demonstrated that the in vivo effect was not mediated by the TDP-43-DSE interaction. In fact, substitution of the Add2 DSE with viral E-SV40 and L-SV40 DSEs, which are not TDP-43 targets, still resulted in decreased Add2 mRNA levels after TDP-43 downregulation. In addition, we failed to show interaction between TDP-43 and key polyadenylation factors, such as CstF-64 and CPSF160 and excluded TDP-43 involvement in pre-mRNA cleavage and regulation of polyA tail length. These evidences allowed us to exclude the pre-hypothesized role of TDP43 in modulating 3 end processing of Add2 pre-mRNA. Finally, we showed that TDP-43 regulates Add2 gene expression levels by increasing Add2 mRNA stability. Considering that Add2 in brain participates in synapse assembly, synaptic plasticity and their stability, and its genetic inactivation in mice leads to LTP, LTD, learning and motor-coordination deficits, we hypothesize that a possible loss of Add2 function by TDP-43 depletion may contribute to ALS and FTD disease states.