Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jan 3;289(1):112-21.
doi: 10.1074/jbc.M113.474700. Epub 2013 Nov 25.

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

Chongtae Kim  1 , 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
Affiliations

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

Chongtae Kim et al. J Biol Chem. .

Erratum in

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 HuD-expressing plasmid. The expression 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 from WT and HuD-null mice. We identified ATG5 mRNA as a post-transcriptional target of the mammalian RNA-binding protein HuD in pancreatic β 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 β cells. Our results reveal HuD to be an inducer of ATG5 expression and hence a critical regulator of autophagosome formation in pancreatic β cells.

Keywords: Autophagy; RNA Metabolism; RNA Turnover; RNA-Protein Interaction; RNA-binding Protein.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
HuD associates to ATG5 mRNA. A, βTC6 cell lysates were subjected to ribonucleoprotein complex immunoprecipitation followed by RT-qPCR analysis to measure the enrichment of ATG5 mRNA in HuD IP compared with control IgG IP. All mRNA amounts were normalized to the levels of GAPDH mRNA (2−[Ct(ATG5 or Ins2)-Ct(GAPDH)]). The data represent means ± S.E. from three independent experiments. *, p < 0.05 B, top, schematic description of the ATG5 mRNA 5′-UTR (5U), coding region (CR), and 3′-UTR (3U1 and 3U2), as well as the biotinylated transcripts synthesized for biotin pulldown analysis. Bottom, each biotinylated transcript was incubated with βTC6 cell lysates, and the interaction between RNA-binding proteins and biotinylated transcripts was analyzed by Western blot analysis using HuD, HuR, AUF1, hnRNPL, TIAR, and NF90 antibodies. C, biotinylated subdivided regions of ATG5 3U2 were synthesized, and fragments A, B, and C were further incubated with βTC6 cell lysates. The interaction between HuD and biotinylated transcripts was analyzed by Western blot analysis using HuD antibody. nt, nucleotide.
FIGURE 2.
FIGURE 2.
HuD regulates ATG5 expression through its 3′-UTR. A, schematic of reporter plasmids: parent vector (pEGFP), a plasmid expressing only one copy of fragment ATG5 3′-UTR1 (pATG5 3U1), and a plasmid expressing only one copy of fragment ATG5 3′-UTR2 (pATG5 3U2). B, 48 h after transfection of either siCtrl or siHuD or 24 h after transfection of either pcDNA or pHuD, together with each reporter plasmids, the levels of EGFP mRNAs were analyzed by RT-qPCR. All mRNA amounts were normalized to the levels of GAPDH mRNA (2−[Ct(EGFP)-Ct(GAPDH)]). The data represent means ± S.E. from three independent experiments. C and D, 48 h after transfection of either siCtrl or siHuD or 24 h after transfection of either pcDNA or pHuD, reporter plasmids (pATG5 3U2 for C; pATG5 3U1 for D) were sequentially transfected. The level of EGFP, HuD, and loading control β-actin were assessed by Western blot analysis. Relative density of the indicated proteins from three independent experiments is shown on the bottom. E, 24 h after transfection of pcDNA or pHuD, miR-181b or control miRNA was transfected into HCT-116 cells, followed by transfection with reporter plasmids (pATG5 3U2 or pEGFP). The expression levels of EGFP and β-actin were analyzed by Western blot analysis.
FIGURE 3.
FIGURE 3.
HuD enhances ATG5 expression. A, 48 h after transfection into βTC6 cells, total RNA was isolated, and the levels of ATG5 mRNA were measured by RT-qPCR. All mRNA amounts were normalized to the levels of GAPDH mRNA (2−[Ct(ATG5)-Ct(GAPDH)]). Left, Ctrl siRNA (siCtrl) versus HuD siRNA (siHuD); right, pcDNA control versus HuD overexpression (pHuD). B, lysates from βTC6 cells that were transfected as described in A were prepared to assess the level of ATG5, HuD, and loading control β-actin by Western blotting. Relative density for the indicated proteins is shown on the bottom. The data represent means ± S.E. from three independent experiments. **, p < 0.01. C and D, 48 h after transfection of siCtrl or siHuD or 24 h after transfection of pcDNA or pHuD, lysates were fractionated through sucrose gradients (C), and the relative distribution of ATG5 mRNA and GAPDH mRNA in each 12 fractions was studied by RT-qPCR analysis (D). 40S, small ribosome subunits; 60S, large ribosome subunits; 80S, mono-ribosome; Polysomes, polyribosome. The data are representative of three independent experiments. E, relative ATG5 mRNA distributions between free subunits/monosomes (fractions 1–6) and polysomes (fractions 7–11) were represented from three independent experiments. *, p < 0.05. F, after co-transfection of reporter construct containing ATG5 3′-UTR (pATG5 3U2) in siHuD or pHuD transfected cells, the distribution of EGFP mRNA and GAPDH mRNA was analyzed as described in D. G, nascent EGFP production was monitored by a brief (20-min-long) incubation with l-[35S]methionine and l-[35S]cysteine 48 h after transfection of siCtrl or siHuD, or 24 h after transfection of pcDNA or pHuD. Lysates were prepared and subjected to IP using anti-EGFP or anti-GAPDH antibodies, and the incorporation of radiolabeled amino acids into newly synthesized EGFP and GAPDH proteins was assessed by SDS-PAGE and visualized using a PharoseFX Plus.
FIGURE 4.
FIGURE 4.
HuD enhances autophagosome formation. A, 48 h after transfection of siRNA (siCtrl or siHuD) or 24 h after transfection of plasmids (pcDNA or pHuD) into βTC6 cells, the levels of HuD, LC3, and loading control β-actin were assessed by Western blot analysis. Relative density for the indicated proteins is shown on the bottom. The data represent the means ± S.E. from three independent experiments. B, U2OS cells stably expressing GFP-LC3 were incubated with 200 μm AICAR or 0.4 μg/ml colchicine for 16 h after transfection with pcDNA or pHuD. The levels of HuD, GFP-LC3, and loading control β-actin were analyzed by Western blot analysis. Relative density for the indicated proteins is shown on the bottom. The data represent the means ± S.E. from three independent experiments. C, 24 h after transfection of pcDNA or pHuD, U2OS/GFP-LC3 cells were incubated with 200 μm AICAR or 0.4 μg/ml colchicine for 16 h and examined under fluorescence microscope. D, the ratio of GFP-LC3 punctated cells to total GFP-positive cells was analyzed under a fluorescence microscope. The data represent the means ± S.E. of three independent experiments. E, 48 h after transfection of siCtrl or siHuD, βTC6 cells were fixed overnight and subjected to transmission electron microscopic analysis. The arrows indicate autophagosomes.
FIGURE 5.
FIGURE 5.
Regulation of autophagosome formation by HuD is ATG5 dependent. 48 h after transfection of siRNAs (siCtrl or siATG5), U2OS/GFP-LC3 cells were transfected with pcDNA or pHuD for 24 h and then treated with 200 μm AICAR (A) or 0.4 μg/ml colchicine (B) for an additional 16 h. The levels of ATG5, HuD, GFP-LC3, and loading control β-actin were assessed by Western blot analysis. Relative density for the indicated proteins is shown on the bottom. The data are representative of three independent experiments.
FIGURE 6.
FIGURE 6.
HuD-null mice show abnormal expression of ATG5 and LC3. The expression level of ATG5 (A) and LC3 (B) were analyzed by immunostaining in pancreatic islets of WT and HuD-null mice. The slides were stained with antibodies against insulin (green) and ATG5 (red) or LC3 (red). Scale bars, 50 μm. For graph, multiple sections from three mice per genotype, separated by at least 200 μm from each section, were imaged, and the relative signal intensity for the indicated proteins was assessed using Image Browser software (Carl Zeiss). *, p < 0.05.
See this image and copyright information in PMC

References

    1. Levine B., Kroemer G. (2008) Autophagy in the pathogenesis of disease. Cell 132, 27–42 - PMC - PubMed
    1. Levine B., Mizushima N., Virgin H. W. (2011) Autophagy in immunity and inflammation. Nature 469, 323–335 - PMC - PubMed
    1. Florey O., Overholtzer M. (2012) Autophagy proteins in macroendocytic engulfment. Trends Cell Biol. 22, 374–380 - PMC - PubMed
    1. Masini M., Bugliani M., Lupi R., del Guerra S., Boggi U., Filipponi F., Marselli L., Masiello P., Marchetti P. (2009) Autophagy in human type 2 diabetes pancreatic beta cells. Diabetologia 52, 1083–1086 - PubMed
    1. Tanida I. (2011) Autophagy basics. Microbiol. Immunol. 55, 1–11 - PubMed

Publication types

MeSH terms