The RNA-binding protein HuD regulates autophagosome formation in pancreatic ß cells by promoting autophagy-related gene 5 expression.

ABSTRACT

Tight regulation of autophagy is critical for the fate of pancreatic ß cells. The autophagy protein ATG5 is essential for the formation of autophagosomes by promoting the lipidation of microtubule-associated protein LC3 (light chain 3). However, little is known about the mechanisms that regulate ATG5 expression levels. In this study, we investigated the regulation of ATG5 expression by HuD. The association of HuD with ATG5 mRNA was analyzed by ribonucleoprotein complex immunoprecipitation and biotin pulldown assays. HuD expression levels in pancreatic ß cells were knocked down via siRNA, elevated by overexpression of a HuD-expressing plasmid. The expression levels of HuD, ATG5, LC3, and ß-actin were determined by Western blot and quantitative RT-PCR analysis. Autophagosome formation was assessed by fluorescence microscopy in GFP-LC3-expressing cells and in pancreatic tissues from WT and HuD-null mice. We identified ATG5 mRNA as a post-transcriptional target of the mammalian RNA-binding protein HuD in pancreatic ß cells. HuD associated with the 3'-UTR of the ATG5 mRNA. Modulating HuD abundance did not alter ATG5 mRNA levels, but HuD silencing decreased ATG5 mRNA translation, and, conversely, HuD overexpression enhanced ATG5 mRNA translation. Through its effect on ATG5, HuD contributed to the lipidation of LC3 and the formation of LC3-positive autophagosomes. In keeping with this regulatory paradigm, HuD-null mice displayed lower ATG5 and LC3 levels in pancreatic ß cells. Our results reveal HuD to be an inducer of ATG5 expression and hence a critical regulator of autophagosome formation in pancreatic ß cells.