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

Research output: Contribution to journalArticle

18 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

Fingerprint

RNA-Binding Proteins
Autophagy
Genes
Gene Expression
Light
Messenger RNA
Protein Biosynthesis
Ribonucleoproteins
Microtubule-Associated Proteins
Fluorescence microscopy
Biotin
Fluorescence Microscopy
Immunoprecipitation
Small Interfering RNA
Autophagosomes
Actins
Assays
Plasmids
Western Blotting
Association reactions

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

The RNA-binding protein HuD regulates autophagosome formation in pancreatic β cells by promoting autophagy-related gene 5 expression. / Kim, Chongtae; Kim, Wook; Lee, Heejin; Ji, Eunbyul; Choe, Yun Jeong; Martindale, Jennifer L.; Akamatsu, Wado; Okano, Hideyuki; Kim, Ho Shik; Nam, Suk Woo; Gorospe, Myriam; Lee, Eun Kyung.

In: Journal of Biological Chemistry, Vol. 289, No. 1, 03.01.2014, p. 112-121.

Research output: Contribution to journalArticle

Kim, C, Kim, W, Lee, H, Ji, E, Choe, YJ, Martindale, JL, Akamatsu, W, Okano, H, Kim, HS, Nam, SW, Gorospe, M & Lee, EK 2014, 'The RNA-binding protein HuD regulates autophagosome formation in pancreatic β cells by promoting autophagy-related gene 5 expression', Journal of Biological Chemistry, vol. 289, no. 1, pp. 112-121. https://doi.org/10.1074/jbc.M113.474700
Kim, Chongtae ; Kim, Wook ; Lee, Heejin ; Ji, Eunbyul ; Choe, Yun Jeong ; Martindale, Jennifer L. ; Akamatsu, Wado ; Okano, Hideyuki ; Kim, Ho Shik ; Nam, Suk Woo ; Gorospe, Myriam ; Lee, Eun Kyung. / The RNA-binding protein HuD regulates autophagosome formation in pancreatic β cells by promoting autophagy-related gene 5 expression. In: Journal of Biological Chemistry. 2014 ; Vol. 289, No. 1. pp. 112-121.
@article{3bd84527d92e4aa5b7c3eee9a69e6007,
title = "The RNA-binding protein HuD regulates autophagosome formation in pancreatic β cells by promoting autophagy-related gene 5 expression",
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.",
author = "Chongtae Kim and Wook Kim and Heejin Lee and Eunbyul Ji and Choe, {Yun Jeong} and Martindale, {Jennifer L.} and Wado Akamatsu and Hideyuki Okano and Kim, {Ho Shik} and Nam, {Suk Woo} and Myriam Gorospe and Lee, {Eun Kyung}",
year = "2014",
month = "1",
day = "3",
doi = "10.1074/jbc.M113.474700",
language = "English",
volume = "289",
pages = "112--121",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "1",

}

TY - JOUR

T1 - The RNA-binding protein HuD regulates autophagosome formation in pancreatic β cells by promoting autophagy-related gene 5 expression

AU - Kim, Chongtae

AU - Kim, Wook

AU - Lee, Heejin

AU - Ji, Eunbyul

AU - Choe, Yun Jeong

AU - Martindale, Jennifer L.

AU - Akamatsu, Wado

AU - Okano, Hideyuki

AU - Kim, Ho Shik

AU - Nam, Suk Woo

AU - Gorospe, Myriam

AU - Lee, Eun Kyung

PY - 2014/1/3

Y1 - 2014/1/3

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84891673895&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84891673895&partnerID=8YFLogxK

U2 - 10.1074/jbc.M113.474700

DO - 10.1074/jbc.M113.474700

M3 - Article

C2 - 24275661

AN - SCOPUS:84891673895

VL - 289

SP - 112

EP - 121

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 1

ER -