Cross regulation of RNA Binding Proteins and Ordered Protein Aggregation

点击翻译按钮获取中文摘要

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.