Mammalian ataxin-2 modulates translation control at the pre-initiation complex via PI3K/mTOR and is induced by starvation - PubMed (original) (raw)

Mammalian ataxin-2 modulates translation control at the pre-initiation complex via PI3K/mTOR and is induced by starvation

Isabel Lastres-Becker et al. Biochim Biophys Acta. 2016 Sep.

Abstract

Ataxin-2 is a cytoplasmic protein, product of the ATXN2 gene, whose deficiency leads to obesity, while its gain-of-function leads to neural atrophy. Ataxin-2 affects RNA homeostasis, but its effects are unclear. Here, immunofluorescence analysis suggested that ataxin-2 associates with 48S pre-initiation components at stress granules in neurons and mouse embryonic fibroblasts, but is not essential for stress granule formation. Coimmunoprecipitation analysis showed associations of ataxin-2 with initiation factors, which were concentrated at monosome fractions of polysome gradients like ataxin-2, unlike its known interactor PABP. Mouse embryonic fibroblasts lacking ataxin-2 showed increased phosphorylation of translation modulators 4E-BP1 and ribosomal protein S6 through the PI3K-mTOR pathways. Indeed, human neuroblastoma cells after trophic deprivation showed a strong induction of ATXN2 transcript via mTOR inhibition. Our results support the notion that ataxin-2 is a nutritional stress-inducible modulator of mRNA translation at the pre-initiation complex.

Keywords: Amyotrophic lateral sclerosis; Diabetes mellitus; Spinocerebellar Ataxia type 2; TORC1; mRNA translation.

Copyright © 2016 Elsevier B.V. All rights reserved.

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Figures

Figure 1

Figure 1. Ataxin-2 colocalizes with TIA-1, PABP, RPS6, eIF4G and eIF4A1 in SG of hippocampal neuron cultures

Hippocampal neurons after 7 days of culture (DIV7) were either left untreated (Control) or were stressed (ARS for arsenite, 0.5 mM for 45 min). The expression and subcellular localization of TIA-1 (A, F), PABP (B, F), RPS6 (C), eIF4G (D), eIF4A1 (E), and ataxin-2 (A-E) was monitored by immunofluorescence. Where indicated, the colocalization of two proteins was studied (Merge). DAPI staining was included to visualize the nuclei.

Figure 1

Figure 1. Ataxin-2 colocalizes with TIA-1, PABP, RPS6, eIF4G and eIF4A1 in SG of hippocampal neuron cultures

Hippocampal neurons after 7 days of culture (DIV7) were either left untreated (Control) or were stressed (ARS for arsenite, 0.5 mM for 45 min). The expression and subcellular localization of TIA-1 (A, F), PABP (B, F), RPS6 (C), eIF4G (D), eIF4A1 (E), and ataxin-2 (A-E) was monitored by immunofluorescence. Where indicated, the colocalization of two proteins was studied (Merge). DAPI staining was included to visualize the nuclei.

Figure 2

Figure 2. The absence of ataxin-2 in MEFs does not impair TIA-1 redistribution from nucleus to cytosolic SG

MEFs from WT and KO were either left untreated (Control) or were stressed (ARS for arsenite, 0.5 mM for 45 min). The subcellular localization of TIA-1 was monitored by immunofluorescence. Where indicated, the colocalization of two proteins was studied (Merge). DAPI staining was included to visualize the nuclei.

Figure 3

Figure 3. The absence of ataxin-2 does not prevent the formation of SG

MEFs from WT and KO were either left untreated (Control) or were stressed (ARS for arsenite, 0.5 mM for 45 min). The subcellular localization of PABP (A), RPS6 (B), eIF4G (C, E), eIF4A1 (D, E), and ataxin-2 (A-D) was monitored by immunofluorescence. Where indicated, the colocalization of two proteins was studied (Merge). DAPI staining was included to visualize the nuclei.

Figure 3

Figure 3. The absence of ataxin-2 does not prevent the formation of SG

MEFs from WT and KO were either left untreated (Control) or were stressed (ARS for arsenite, 0.5 mM for 45 min). The subcellular localization of PABP (A), RPS6 (B), eIF4G (C, E), eIF4A1 (D, E), and ataxin-2 (A-D) was monitored by immunofluorescence. Where indicated, the colocalization of two proteins was studied (Merge). DAPI staining was included to visualize the nuclei.

Figure 3

Figure 3. The absence of ataxin-2 does not prevent the formation of SG

MEFs from WT and KO were either left untreated (Control) or were stressed (ARS for arsenite, 0.5 mM for 45 min). The subcellular localization of PABP (A), RPS6 (B), eIF4G (C, E), eIF4A1 (D, E), and ataxin-2 (A-D) was monitored by immunofluorescence. Where indicated, the colocalization of two proteins was studied (Merge). DAPI staining was included to visualize the nuclei.

Figure 4

Figure 4. Ataxin-2 coimmunoprecipitates with TIA-1 and the translation pre-initiation complex

HEK-293 cells were left untreated or stressed with 0.5 mM arsenite for 45 min. Ataxin-2 was immunoprecipitated from cell homogenates with the 12SCA2-2B6 monoclonal antibody against ataxin-2 (IP ATXN2). Immunoprecipitates were analyzed by PAGE and western blotting with detection by the relevant antibodies. (A) Similar amounts of TIA-1 were coimmunoprecipitated with ataxin-2 in unstressed and stressed cells. (B) Similar amounts of several constituents of the translation pre-initiation complex were coimmunoprecipitated with ataxin-2 in unstressed and stressed cells.

Figure 5

Figure 5. Polysome gradients reveal ataxin-2 distribution and KO effects

(A, B) Polysome profiles on sucrose gradients: Lysates from WT-MEFs (A) and KO-MEFs (B) were layered on a 10-ml continuous sucrose gradient (10% to 50% sucrose). The profile of optical density representing the nucleotide content of the 40S, 60S, 80S ribosome components and polysomes are indicated. (C, D) Western blot analyses of the distribution of ataxin-2 and pre-initiation complex protein components among fractions from polysomal profiles in WT-MEFs (C) and KO-MEFs (D).

Figure 6

Figure 6. The absence of ataxin-2 induces increased phosphorylation of the ribosomal protein S6 and of the translation initiation repressor 4E-BP1, mainly through activation of the PI3K and the mTOR pathway

Phosphorylation of RPS6 (A) and 4E-BP1 (B) in WT- and KO-MEFs after 24 h of serum deprivation was measured using an ELISA kit, with the graphs illustrating the ratio of phospho-RPS6 versus total-RPS6 levels and of phospho-4E-BP1 versus total 4E-BP1. (C-D): Thirty min before cell harvest, MEF cultures were treated with the MEK inhibitor U0126, the PI3K inhibitor LY294002 or the mTOR inhibitor rapamycin; 5 WT- and 5 KO-MEF lines were tested in each case for RPS6 phosphorylation (C) and 4E-BP1 phosphorylation (D), with each experiment being performed three times (* p<0.05, ** p<0.01, *** p<0.005; * significant between WT-MEFs and KO-MEFs; # significant between KO-MEF different treatments). The relative results are presented with arbitrary units.

Figure 7

Figure 7. Nutrient and serum starvation elicits a transcriptional induction of ATXN2 via the mTOR pathway

(A) Human SH-SY5Y neuroblastoma cells were cultured in RPMI medium containing 10% FCS (RPMI + FCS) or in HBSS without nutrients (HBSS - FCS). At the indicated time points the ATXN2 transcript levels were quantified by qPCR and normalized to non-starvation (RPMI + FCS) at time 2 h. Student's t-test was used to calculate significance between starved and non-starved cells. (B) SH-SY5Y cells either treated with control (RPMI + FCS, white bars) or starvation medium (HBSS – FCS, black bars) with 0.05% DMSO, 5 nM bafilomycin A1 (BAF), 0.5 μM rapamycin as mTOR pathway antagonist (RAP), 25 μM LY294002 (LY) or nothing (CON) were incubated for 16 h prior to mRNA analysis by qPCR (* p<0.05, ** p<0.01, *** p<0.005).

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