Poster abstracts

Poster number 116 submitted by Erik Koppes

Ablation of the imprinted PWS-orthologous domain in a rat insulinoma cell line reveals cell-autonomous transcriptional deficiencies in peptide hormones and ER chaperones independent of ER stress

Erik A. Koppes (Department of Pediatrics, Division of Genetic & Genomic Medicine, School of Medicine, University of Pittsburgh), Marie A. Johnson (Department of Pediatrics, Division of Genetic & Genomic Medicine, School of Medicine, University of Pittsburgh), Hyun J. Park (Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh), Peter Drain (Department of Cell Biology, School of Medicine, University of Pittsburgh), Robert D. Nicholls ( Department of Pediatrics, Division of Genetic & Genomic Medicine, School of Medicine, University of Pittsburgh)

The Prader-Willi syndrome (PWS) imprinted domain spans multiple protein-coding and non-coding paternally expressed genes, including extended lncRNAs encompassing bicistronic SNURF-SNRPN and five classes of box-C/D snoRNAs. Characteristic PWS clinical features of neonatal hypotonia, juvenile onset hyperphagia and obesity, hormone deficiencies, and additional comorbidities manifest when this domain is paternally deleted or bimaternally inherited. Previously, we established a neonatally lethal hypoglycemia phenotype subsequent to hypoinsulinemia and hypoglucagonemia in a PWS mouse model (1, 2). To delineate pathophysiological mechanisms, we engineered a 3-Mb deletion of the PWS-orthologous domain in rat INS-1 cells and identified cell-autonomous deficits in basal and glucose-stimulated insulin secretion (3). Transcriptomic analysis by RNA-Seq revealed decreased expression of peptide hormones Iapp, Npy and an Insulin::mCherry transgene and for numerous ER-chaperones, critical to their production. These changes were validated by congruent proteomic and targeted RT-ddPCR and western blot experiments. While PWS INS-1 lines had a propensity for a more robust early chemically induced activation of ER-stress pathways, including IRE1α/XBP1, ATF6 and eIF2α, there was no basal activation of ER-stress or the unfolded protein response. Additionally, both control and PWS β-cells similarly activated profound ER-stress transcriptome-wide changes at 5 hours of thapsigargin treatment and had widespread changes in alternative splicing. Genes with robust activation on ER-stress in INS-1 cells included Atf3, Trib3, Ddit3 and Rcn1. Intriguingly, differential RNA-splicing or intron-retention events occurred for the Snrpn ancestral paralog Snrpb, the SR-protein splicing factor Srsf3, and multiple ribosomal protein genes with embedded snoRNA genes. Current studies are further assessing molecular events underlying transcriptional alterations in PWS β-cells and in β-cells under ER stress.

1. Stefan M, et al. (2005) Hormonal and metabolic defects in a Prader-Willi syndrome mouse model with neonatal failure to thrive. Endocrinology 146, 4377-85

2. Stefan M, et al. (2011) Global deficits in development, function, and gene expression in the endocrine pancreas in a deletion mouse model of Prader-Willi syndrome. AJP-Endo 300, E909-22

3. Koppes EA, et al. (2022) Insulin secretion deficits in a Prader-Willi syndrome β-cell model are associated with a concerted downregulation of multiple endoplasmic reticulum chaperones.

Keywords: RNA-Seq, ncRNA, Alternative Splicing