Investigating the role of Pdx1 in coordinating translational regulation in pancreatic beta cells

University Of Pennsylvania, Philadelphia PA

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Abstract

? DESCRIPTION (provided by applicant): Pancreatic ß cell dysfunction plays a central role in the development and progression of type 2 diabetes (T2D) as this leads to inadequate insulin release to compensate for insulin resistance of peripheral tissues. The transcription factor Pdx1 is critical for maintaining ß cell identity and function, and mutations in this factor lead to diaetes in both humans and mice. Therefore, understanding the downstream regulatory networks established by this factor will uncover pathways that are essential for ß cell function. Importantly, Pdx1 may shape gene expression in ß cells not only at the level of transcription, but also by controlling the expression of factors which shape the translational landscape of these cells. The goal of this proposal is to determine the role of Pdx1 in shaping translational regulation in ß cells and the relevance of this regulation for the proper response to cellular stresses that are associated with T2D. Translational regulation is especially pervasive during ER stress, a condition proposed to contribute to ß cell dysfunction in T2D. Pdx1 is critical for te proper ER stress response in ß cells, but the degree to which Pdx1 may coordinate translational regulation during ER stress is unknown. Further, a preliminary screen indicates that Pdx1 deficiency does in fact cause a change in translational efficiency for approximately 200 transcripts under basal conditions, including factors involved in maintenance of ß cell identity and response to oxidative stress. For example, the transcription factor Nkx2.2 has increased translational efficiency in the context of Pdx1 deficiency, which is particularly interesting because this factor suppresses ß cell reprogramming. In this proposal, translational regulation will be studied on a genome-wide scale by using the translating ribosome affinity purification (TRAP) methodology, which involves immunoprecipitation of a tagged ribosomal protein to isolate ribosome-associated RNA. This approach also has the distinct advantage of allowing for the determination of ribosome occupancy, which approximates translational efficiency, of transcripts in a specific cell type within a heterogeneous tissue. First, this method will be used n both a ß cell line and a mouse model to study the effect of Pdx1 deficiency on translational regulation during ER stress. The second focus of this proposal is to investigate the mechanism underlying the Pdx1-dependent translational regulation observed in the preliminary TRAP screen. De novo motif discovery on the transcripts from this screen has implicated a cytosine-rich motif in Pdx1-dependent translational control over many of these transcripts, including Nxk2.2. A reporter assay will be used to investigate the importance of this motif for translational regulation of the Nkx2.2 mRNA. RNA-pull down assays will then identify potential RNA binding proteins interacting with this motif. Together, the proposed experiments will improve our understanding of Pdx1-dependent translational regulation in pancreatic ß cells and the relevance of this regulation during cellular stresses.