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. 2013 Apr 1;1(1):e23980.
doi: 10.4161/trla.23980.

HuR controls mitochondrial morphology through the regulation of BclxL translation

Danielle Durie  1 , Maria Hatzoglou  2 , Pranesh Chakraborty  3 , Martin Holcik  4
Affiliations

HuR controls mitochondrial morphology through the regulation of BclxL translation (VSports注册入口)

Danielle Durie et al. Translation (Austin). .

VSports - Abstract

BclxL is a key prosurvival factor that in addition to controlling mitochondrial membrane permeability regulates mitochondrial network dynamics. The expression of BclxL is regulated at the level of translation, splicing and selective translation VSports手机版. In this study, we show that the RNA-binding protein HuR, which is known to orchestrate an anti-apoptotic cellular program, functions as a translational repressor of BclxL. We show that HuR binds directly to the 5'UTR of BclxL, and represses BclxL translation through the inhibition of its internal ribosome entry site (IRES). Reduction of HuR levels leads to the derepression of BclxL translation and subsequent rearrangement of the mitochondrial network. Our results place BclxL into the HuR-regulated operon and provide further insight into the regulation of cellular stress response by HuR. .

Keywords: IRES; ITAF; RNA binding protein; mitochondrial dynamics; translational control V体育安卓版. .

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Figures

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Figure 1. Cellular levels of HuR control the expression of BclxL. U2OS cells were transiently transfected with HuR-targeting or non-silencing (Ctrl) siRNA and the levels of BclxL (A), or Bcl-2 and Mcl-1 (B) were determined 72 h after transfection by western blot analysis. Signal intensities were quantified by densitometry and are shown on the right (fold). (C) U2OS cells were transiently transfected with HuR-targeting or non-silencing (Ctrl) siRNA, and 48 h later transfected with GFP- or GFP-HuR expressing plasmid as indicated. The expression levels of BclxL were determined 24 h following the plasmid transfection by western blot analysis (* denotes GFP-HuR). Signal intensities were quantified by densitometry and are shown in (D). (* p < 0.05; n = 3).
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Figure 2. HuR regulates translation of BclxL. U2OS cells were transiently transfected with HuR-targeting or non-silencing (Ctrl) siRNA, or with GFP- or GFP-HuR expressing plasmid and the steady-state levels of Bclx (A) and BclxL (B) mRNAs were determined by quantitative RT-PCR. (C) Splice-variant specific RT-PCR was performed on RNA extracted from U2OS cells transiently transfected with HuR-targeting or non-silencing (Ctrl) siRNA. The position of BclxL and BclxS is indicated on the right. (D) Representative polysome profile trace of HEK293 cells transiently transfected with HuR-targeting or non-silencing (Ctrl) siRNA. Fractions (0 – top; 10 – bottom) are indicated below the graph. (E) Analysis of de novo proteins synthesis by L-[35S]methionine labeling of HEK293 cells after transfection with either HuR-targeting or non-silencing (Ctrl) siRNA. GAPDH was immunoprecipitated using anti-GAPDH antibodies. (F) The relative polysome abundance of BclxL mRNA calculated from polysome profiling (monosomes – fractions 1–4; polysomes – fractions 5–9).
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Figure 3. HuR binds directly to the 5′ UTR of BclxL and regulates its translation. (A) Schematic diagram of BclxL 5′ UTR and the deletion fragments used in the binding assays. (B) Recombinant GST-HuR was incubated in the presence of 32P-labeled, in vitro transcribed RNA (probe A) and subjected to UV cross-linking. RNA-protein complexes were separated by SDS-PAGE and analyzed by autoradiography. GST was used as a negative control. (C) Increasing concentrations of GST-HuR were incubated with indicated, 32P-labeled, in vitro transcribed RNAs and nitrocellulose filter binding assays were performed as described in Material and Methods. Levels of filter-bound RNA are plotted as a function of protein concentration. Apparent dissociation constants (Kd) are shown for each probe (mean +/− S.E.M., n = 3). (D) Bicistronic DNA construct containing BclxL 5′ UTR (pßgal/BclxL/CAT), or a parental vector (pßgal/CAT) were co-transfected into HEK293 cells along with GFP- or GFP-HuR expressing plasmids and the expression levels of each reporter gene were determined 24 h after transfection (** p < 0.01; n = 3).
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Figure 4. HuR regulates mitochondrial morphology through BclxL. (A) U2OS were transiently transfected with HuR-targeting, non-silencing (Ctrl), or a combination of HuR- and BclxL -targeting siRNA and the cells were prepared for immunofluorescent microscopy 72 h later. Mitochondrial morphology was visualized by staining with anti-Tom20 antibody. (B) The fraction of cells with fragmented mitochondria from (A) were determined by two blind observers and is plotted for each treatment (* p < 0.05; n = 83 cells for each treatment) (C) Expression levels of BclxL and HuR in lysates from cells treated as in (A) were determined by western blot analysis.
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