? cell dysfunction and ? cell failure play central roles in the pathogenesis of Type 2 Diabetes Mellitus (T2DM). Increasing evidence has suggested that post-transcriptional regulation of gene expression is essential for ? cell function and survival. However, the underlying mechanisms governing how the post-transcriptional landscape is shaped to promote ? homeostasis remains largely unexplored. A growing body of evidence has implicated RNA binding proteins (RBPs) in influencing the metabolism of mRNAs that encode products essential for ? cell survival. Despite these findings, few RBPs have been examined in understanding how the post-transcriptional milieu is shaped to maintain ? cell homeostasis. Based on a Translating Ribosomal Affinity Purification (TRAP) screen in our laboratory using the Min6 ? cell line, the poly(C) binding protein family of RNA binding proteins, specifically PCBP1 and PCBP1, have been raised as potential post-transcriptional regulators of ? cell identity and survival genes. Thus, the goal of this proposal is to elucidate the mechanistic and physiological importance of post-transcriptional regulation by PCBP1 and PCBP2 in ? cells. In the first aim of this proposal, the in vivo roles exerted by PCBP1 and PCBP2 will be examined by crossing mice harboring conditional alleles for Pcbp1 and Pcbp2 with a ? cell specific Cre deleter strain. Morphological and functional analyses will be performed with ? cells that have individual and combined Pcbp1/2 deficiency. Further, the inclusion of a lineage trace allele will allow us to determine the fate of Pcbp1/2 deficient ? cells. The second part of this proposal will examine how glucose shapes the post-transcriptional landscape of ? cells. ? cells are highly regulated by glucose to allow for tight regulation of blood glucose homeostasis. Interestingly, preliminary demonstrate that Pcbp2 expression substantially increases in murine and human islets exposed to high glucose, pointing to a role in PCBP2 post-transcriptionally mediating the effects of glucose in the ? cell. To determine the influence of glucose on the post-transcriptional landscape and the role of PCBP2 in this response, TRAP will be used in wild-type and Pcbp2-deficient ? cells exposed to low and high glucose. TRAP has the distinct advantage of determining ribosomal occupancy, or translational efficiency, specifically in ? cells within isolated pancreatic islets, which are a heterogenous tissue type. At the same time, analysis of total RNA from isolated ? cells will reveal to what extent PCBP2 impacts other aspects of post-transcriptional gene regulation affected by elevated glucose. Additionally, Cross-Linking and Immunoprecipitation Sequencing (CLIP-Seq) will be performed in wild-type and Pcbp2-deficient ? cells to identify PCBP2 binding sites transcriptome-wide to determine whether PCBP2 binds to transcripts it functionally impacts. Altogether, these experiments will establish a paradigm for PCBP1 and PCBP2 in preserving ? cell homeostasis and will provide mechanistic insight into post- transcriptional regulation of ? cell gene expression which could be leveraged in efforts to prevent T2DM.
Type 2 Diabetes Mellitus (T2DM) results from failure of the insulin producing ? cell to maintain proper blood glucose homeostasis. While much progress has been made in elucidating the causes of ? cell failure in T2DM pathogenesis, mechanisms employed by ? cells to promote cellular homeostasis remain less-well defined. We have identified novel factors that impact how ? cells preserve their function and identity and the data generated from examining these factors could be leveraged to aid therapeutic development to defend against ? cell failure and to prevent the onset of T2DM.
