Poly(A) tail length and PABP-dependent inhibition of translation by hnRNP-Q2 in Krebs extract.

<p>(A) Krebs extract that was not nuclease-treated was programmed with Cap-Luc mRNAs (0.2 µg/ml) bearing poly(A) tails of the indicated length. Control buffer of hnRNP-Q2 (20 µg/ml) was added to the reaction mixtures as indicated. (B) Sequestering of PABP by Paip2 renders translation insensitive to the poly(A) tail length and inhibition by hnRNP-Q2. Cap-Luc mRNA with increasing poly(A) tails was translated in the untreated extract in the presence of Paip2 (15 µg/ml) as described for panel A. hnRNP-Q2 (20 µg/ml) was added to the reaction mixtures where indicated. Inhibition of translation by hnRNP-Q2 is shown on the top of the panels. (C) Endogenous [³⁵S]methionine incorporation in the untreated extract in the presence of the indicated concentrations of hnRNP-Q2 or 10 µM hippuristanol (Hipp). Incubation was at 32°C for 2 h. Average values for trichloroacetic acid-insoluble radioactivity in 1-µl aliquots of the samples from three assays with standard deviations are shown.</p

Control of Translation and miRNA-Dependent Repression by a Novel Poly(A) Binding Protein, hnRNP-Q

<div><p>Translation control often operates via remodeling of messenger ribonucleoprotein particles. The poly(A) binding protein (PABP) simultaneously interacts with the 3′ poly(A) tail of the mRNA and the eukaryotic translation initiation factor 4G (eIF4G) to stimulate translation. PABP also promotes miRNA-dependent deadenylation and translational repression of target mRNAs. We demonstrate that isoform 2 of the mouse heterogeneous nuclear protein Q (hnRNP-Q2/SYNCRIP) binds poly(A) by default when PABP binding is inhibited. In addition, hnRNP-Q2 competes with PABP for binding to poly(A) in vitro. Depleting hnRNP-Q2 from translation extracts stimulates cap-dependent and IRES-mediated translation that is dependent on the PABP/poly(A) complex. Adding recombinant hnRNP-Q2 to the extracts inhibited translation in a poly(A) tail-dependent manner. The displacement of PABP from the poly(A) tail by hnRNP-Q2 impaired the association of eIF4E with the 5′ m⁷G cap structure of mRNA, resulting in the inhibition of 48S and 80S ribosome initiation complex formation. In mouse fibroblasts, silencing of hnRNP-Q2 stimulated translation. In addition, hnRNP-Q2 impeded let-7a miRNA-mediated deadenylation and repression of target mRNAs, which require PABP. Thus, by competing with PABP, hnRNP-Q2 plays important roles in the regulation of global translation and miRNA-mediated repression of specific mRNAs.</p></div

Detection of poly(A) interacting proteins using UV crosslinking.

<p>RRL (A), HeLa (B), or Krebs (C) S10 cytoplasmic extracts were subjected to UV-induced crosslinking with the ³²P-poly(A) tail. The extracts that were either mock-depleted (Control S10) or depleted of PABP were incubated with ³²P poly(A) tail-labeled globin mRNA at 32°C for 10 min. Prior to adding mRNA, the reaction mixtures were pre-incubated at 32°C for 2 min with GST-Paip2 (20 µg/ml) or PABP (5 µg/ml) as indicated. After UV irradiation and RNase treatment, labeled proteins were analyzed by SDS-PAGE and autoradiography. The positions of molecular mass markers are indicated on the right. Western blotting with an anti-PABP antibody was used to confirm sufficient depletion of PABP from the extracts (for representative analyses, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001564#pbio.1001564-Svitkin2" target="_blank">[54]</a>). Of note, in this and other UV crosslinking analyses, PABP appeared as a fussy band, most probably because RNase digestion does not produce homogenous cross-linked RNA fragments.</p

Additional file 1 of Depletion of LONP2 unmasks differential requirements for peroxisomal function between cell types and in cholesterol metabolism

Additional file 1. Supplementary figures

HnRNP-Q2 inhibits m⁷G cap structure recognition by translation initiation factors.

<p>(A–D) Inhibition of 80S and 48S initiation complex formation by hnRNP-Q2 in nuclease-treated RRL. 80S ribosome binding to 3′ end labeled globin mRNA was assayed in a cycloheximide (0.6 mM)-supplemented RRL, normal (A) or hnRNP-Q2-depleted (B), in the presence of control buffer (squares) or recombinant hnRNP-Q2 (15 µg/ml) (triangles). (C) Validation of the 48S pre-initiation complex formation in the presence of GMPPNP. GTP or GMPPNP were added to the reaction mixtures at 2 mM final concentration as indicated. Other conditions were similar to those described for panel B. (D) 48S pre-initiation complex formation in hnRNP-Q2-depleted RRL in the presence of GMPPNP and either control buffer (squares) or hnRNP-Q2 (25 µg/ml) (triangles). The reaction mixtures were analyzed on 5-ml 15%–30% (A and B) or 11-ml 10%–30% (C and D) sucrose gradients. (E) HnRNP-Q2 dose-dependent inhibition of eIF4E binding to the m⁷G cap structure in RRL as analyzed by chemical crosslinking. Control and hnRNP-Q2-depleted RRL were incubated with oxidized ³²P-cap-labeled poly(A)-extended Luc mRNA in the absence or presence of the indicated concentrations of recombinant hnRNP-Q2. The positions of eIF4E and eIF4A are indicated. Relative efficiencies of eIF4E crosslinking are indicated at the bottom (the value obtained for control RRL was set as 100%).</p

eIF4E controls proliferation in T cell subsets.

<p>(a) Inhibition of eIF4E activity suppresses T_Foxp3− cell proliferation. eFluor 670-labeled T_Foxp3− cells were IL-2/TCR-activated for 72 h in the presence of increasing concentrations of the eIF4E inhibitor 4ei-1 (K_d = 0.80 µM). Proliferation was determined under each condition by eFluor 670 dilution assessed by flow cytometry (upper panel). The effect on proliferation was also assessed by comparing cell counts after 72 h under each condition (lower panel; the control was set to 100%). (b) Inhibition of eIF4E activity abrogates IL-2-mediated reversal of anergy in T_Foxp3+ cells. IL-2/TCR-activated eFluor 670 labelled T_Foxp3+ cells were cultured in the presence of 4ei-1, and proliferation was determined as described in (a). (c–d) IL-2/TCR-activated eFluor 670 labelled T_Foxp3− cells (c) or T_Foxp3+ cells (d) were cultured in the presence of 4ei-1 or 4ei-4. Proliferation was determined under each condition as described in (a). (a–d) Representative histograms from 4 independent experiments are shown (upper panels; the percentages of proliferating cells are indicated). Means and standard deviations of cell counts from 4 independent experiments are shown (lower panel). (e) Induction of T_Foxp3+ cell proliferation occurs independently of signalling through 4E-BPs. 4E-BPdko T_Foxp3+ and T_Foxp3− cells were plated and counted as described in (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003494#pgen-1003494-g005" target="_blank">Figure 5d</a>), and the fold increase in cell number was calculated and associated means and standard deviations (n = 2) are shown. Welch's two sample t-test was used to compare 4E-BPdko T_Foxp3+ cells cultured under different IL-2 concentrations. Also shown is a western blot of total protein extracts probed with antibodies for eIF4E in 4E-BPdko T_Foxp3+ and T_Foxp3− cells. Densitometry was used to quantify protein levels and obtained levels were normalized to β-actin (the normalized values were related to T_Foxp3− 72 h IL-2 100 U/ml which was set to 1 and are indicated above each lane). (f) Ki-67 and eIF4E co-expression in total CD4⁺ T cells isolated directly <i>ex vivo</i> from lymph nodes (left panel). Quantification of eIF4E expression is shown as Δ (eIF4E <i>vs.</i> isotype control) mean fluorescent intensity (MFI). Filled histograms represent staining with an isotype control. Quantification of eIF4E expression (ΔMFI) in Ki-67^+/− T_Foxp3− and T_Foxp3+ cells isolated directly <i>ex vivo</i> (right panel, mean and standard deviation is indicated, n = 3). (g–h) eFluor 670-labeled T_Foxp3− or T_Foxp3+ cells adoptively transferred into separate TCR β−/− mice were isolated from mesenteric (mes) and peripheral (per) lymph nodes (LN) followed by measurement of eFluor 670 and eIF4E expression four days post transfer. (g) Representative dot plots (n = 3) of T_Foxp3− and T_Foxp3+ cell proliferation relative to eIF4E expression in mesLN. Staining with an isotype control are shown as contour plots. (h) Quantification of eIF4E expression (ΔMFI) in cells that have (eFluor 670 low) or have not (eFluor 670 high) undergone cell division (means and standard deviations are indicated after per experiment normalization to T_Foxp3+ cells, n = 4–6). P-value (Welch two sample t-test) is indicated.</p

Inhibition of eIF4E activity results in spontaneous induction of Foxp3 expression in activated T_Foxp3− cells.

<p>T_Foxp3− cells were IL-2/TCR-activated for 72 h in the presence of increasing concentrations of 4ei-1 or the control pro-drug 4ei-4 in undifferentiating conditions, and Foxp3 expression (i.e. GFP) was assessed by flow cytometry. (a) Representative density plots from experiments using T_Foxp3− cells cultured in the presence of 4ei-1 from 4 independent experiments are shown. (b) Percentage Foxp3⁺ cells following treatment with 4ei-1 or 4ei-4 (shown are means and standard deviations, n = 4).</p

Additional file 2 of Depletion of LONP2 unmasks differential requirements for peroxisomal function between cell types and in cholesterol metabolism

Additional file 2. The file contains the following sheets, preceded with 'U2OS' or 'COS-7' : 1. counts_allgenes = raw read counts for all detected genes. 2. counts_analysedgenes = raw not normalised read counts for genes with at least 4 reads in at least 3 samples in the dataset. This gene list was used for differential expression analysis. 3. Deseq2 = differential expression analysis. 4. transformed reads_vsd or transformed reads_rlog = Deseq2 transformation of reads, used for generating heatmaps. 5. counts_normalised = Deseq2 normalisation of reads, used for generating scatter and others plots of indivual gene expression levels. 6. overlay sheets = overlay of COS-7 and U2OS datasets

Distinct modular translational control between activated CD4⁺ T cell subsets.

<p>Graphical representation of the enrichment analysis within subsets of mRNAs identified as differentially expressed (up in T_Foxp3+ cells or down in T_Foxp3+ cells) in data from cytosolic mRNA, polysome-associated mRNA and as differentially translated by anota (after correction for cytosolic mRNA levels). The subsets are shown as columns and the rows represent cellular functions that were enriched. The colour scale represents −log10 p-values (adjusted for multiple testing) for the enrichment. All p-values that were <10e-7 were set to 10e-7.</p

Distinct Translational Control in CD4⁺ T Cell Subsets

<div><p>Regulatory T cells expressing the transcription factor Foxp3 play indispensable roles for the induction and maintenance of immunological self-tolerance and immune homeostasis. Genome-wide mRNA expression studies have defined canonical signatures of T cell subsets. Changes in steady-state mRNA levels, however, often do not reflect those of corresponding proteins due to post-transcriptional mechanisms including mRNA translation. Here, we unveil a unique translational signature, contrasting CD4⁺Foxp3⁺ regulatory T (T_Foxp3+) and CD4⁺Foxp3⁻ non-regulatory T (T_Foxp3−) cells, which imprints subset-specific protein expression. We further show that translation of eukaryotic translation initiation factor 4E (eIF4E) is induced during T cell activation and, in turn, regulates translation of cell cycle related mRNAs and proliferation in both T_Foxp3− and T_Foxp3+ cells. Unexpectedly, eIF4E also affects Foxp3 expression and thereby lineage identity. Thus, mRNA–specific translational control directs both common and distinct cellular processes in CD4⁺ T cell subsets.</p></div