Cross regulation of RNA Binding Proteins and Ordered Protein Aggregation

许多RNA结合蛋白含有一个富含甘氨酸的结构域(GRD)。含有GRD的限制性商业惯例受到严格调控，以避免自发自组装/聚集成细胞质内含物和/或其核耗尽。GRD介导的同型(和异型)蛋白质-蛋白质相互作用可能调节RBP的活性、功能特异性和亚细胞定位。HnRNP A1是一种高度丰富的含有GRD的RBP。它以两种主要的剪接形式存在：A1和A1B，后者是由包含替代外显子7B引起的，该外显子有效地将C-末端GRD扩展了56个残基。虽然hnRNP A1是众所周知的结构性和选择性剪接调节因子，但关于这种较长的异构体的信息很少。最近的一篇论文描述了位于hnRNP A1 GRD内的保守残基(D262N)的一个点突变，它可以驱动hnRNP A1聚集。HnRNP A1聚集的功能结果(S)尚不清楚。*在我们对另一种含有GRD的RBP TDP-43的研究过程中，我们发现了hnRNP A1和TDP-43之间的一种新的关系。TDP-43在RNA代谢中具有广泛的功能。利用siRNA，我们初步确定hnRNP A1受TDP-43转录调控。此外，无细胞实验表明TDP-43也可以直接修改hnRNP A1剪接决定。我们的初步数据表明，TDP-43可以调节hnRNP A1的表达及其选择性剪接，从而突出了hnRNP A1和TDP-43之间意想不到的联系。此外，已发表的RNA-SEQ研究表明，这两个限制性商业惯例共同与大约三分之二的转录组结合(并因此调节)。虽然有几个限制性商业惯例交叉监管的例子，但hnRNP A1和TDP-43之间的联系仍未探索，这是本提案的重点。鉴于这两种限制性商业惯例的丰度相对较高，这一调控的中断预计将对细胞生存产生负面影响。我们假设hnRNP A1受TDP-43的调控，从而控制hnRNP A1B的产生，从而干扰hnRNP A1的正常可逆蛋白质聚集，从而干扰正常的hnRNP A1功能。目前的提案调查了这一调节的性质和细胞/功能后果，重点是蛋白质聚集。未来5年的研究计划的目标是：*1.确定TDP-43调控hnRNP A1/A1B的机制。*2.确定hnRNP A1B中扩展的GRD结构域是否增强了其聚集性。*3.评估hnRNP A1B对剪接和蛋白质相互作用的功能影响。*有序和可逆的蛋白质聚集的概念在许多基本的生物过程中都占有重要地位。鉴于hnRNP A1和TDP-43共同控制着广泛的基因组，这些限制性商业惯例之间的相互作用在基础细胞生物学中可能具有非常重要的意义。

Many RNA binding proteins contain a glycine-rich domain (GRD). GRD-containing RBPs are tightly regulated in order to avoid spontaneous self-assembly/aggregation into cytoplasmic inclusions and/or their nuclear depletion. Homotypic (and heterotypic) protein-protein interactions mediated by the GRD may regulate RBP activity, functional specificity, and subcellular localization. hnRNP A1 is a highly abundant GRD-containing RBP. It exists in two main splice forms: A1 and A1B, the latter arising from the inclusion of an alternative exon 7B which effectively expands the C-terminal GRD by 56 residues. While hnRNP A1 is a well-known regulator of constitutive and alternative splicing, little information is available on this longer isoform. A recent paper describes a point mutation of a conserved residue (D262N) located within the GRD of hnRNP A1 that can drive hnRNP A1 aggregation. The functional consequence(s) of hnRNP A1 aggregation is (are) unknown.*******In the course of our studies of another GRD-containing RBP, TDP-43, we uncovered a novel relationship between hnRNP A1 and TDP-43. TDP-43 functions broadly in RNA metabolism. Using siRNA, we have preliminarily determined that hnRNP A1 is transcriptionally regulated by TDP-43. Moreover, a cell-free assay reveals that TDP-43 can also directly modify hnRNP A1 splice decisions. Our preliminary data indicate that TDP-43 can regulate hnRNP A1 expression as well as its alternative splicing, and thus highlight an unexpected link between hnRNP A1 and TDP-43. Moreover, published RNA-seq studies indicate that these two RBPs collectively bind to (and thus regulate) approximately two-thirds of the transcriptome. While there are several examples of cross-regulation of RBPs, the link between hnRNP A1 and TDP-43 remains unexplored and is the focus of this proposal. Given the relatively high abundance of these two RBPs, disruption of this regulation would be expected to negatively impact cell survival. We hypothesize that hnRNP A1 is regulated by TDP-43, so as to control the production of hnRNP A1B which will interfere with the normal reversible protein aggregation of hnRNP A1 and thus disturb normal hnRNP A1 function. The current proposal investigates the nature of this regulation and the cellular/functional consequences with an emphasis on protein aggregation. The objectives of the research program for the next 5 years are:****1. Determine the mechanism by which hnRNP A1/A1B is regulated by TDP-43.****2. Determine if the extended GRD domain in hnRNP A1B enhances its aggregation.****3. Evaluate the functional impact of hnRNP A1B on splicing and protein interactions.****The concept of ordered and reversible protein aggregation figures prominently in many fundamental biological processes. Given that hnRNP A1 and TDP-43 collectively control a wide swath of the genome, the interplay between these RBPs is potentially highly significant in basic cell biology.***