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

Chongtae Kim, Wook Kim, Heejin Lee, Eunbyul Ji, Yun Jeong Choe, Jennifer L. Martindale, Wado Akamatsu, Hideyuki Okano, Ho Shik Kim, Suk Woo Nam, Myriam Gorospe, Eun Kyung Lee

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21 Citations (Scopus)

Abstract

Tight regulation of autophagy is critical for the fate of pancreatic β 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 β cells were knocked down via siRNA, elevated by overexpression of a HuDexpressing plasmid. Theexpression levels of HuD, ATG5, LC3, and β-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 fromWTand HuD-null mice. We identified ATG5 mRNA as a post-transcriptional target of the mammalian RNAbinding protein HuD in pancreatic β cells. HuD associated with the 3'-UTRof the ATG5m RNA. Modulating Hu Dabun dance 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 βcells. Ourresults revealHuDtobeaninducer of ATG5 expression and hence a critical regulator of autophagosome formation in pancreaticβ cells.

Original languageEnglish
Pages (from-to)112-121
Number of pages10
JournalJournal of Biological Chemistry
Volume289
Issue number1
DOIs
Publication statusPublished - 2014 Jan 3

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Kim, C., Kim, W., Lee, H., Ji, E., Choe, Y. J., Martindale, J. L., Akamatsu, W., Okano, H., Kim, H. S., Nam, S. W., Gorospe, M., & Lee, E. K. (2014). The RNA-binding protein HuD regulates autophagosome formation in pancreatic β cells by promoting autophagy-related gene 5 expression. Journal of Biological Chemistry, 289(1), 112-121. https://doi.org/10.1074/jbc.M113.474700