A Conserved Stem Loop Motif in the 5′Untranslated Region Regulates Transforming Growth Factor-β1 Translation

Transforming growth factor-β1 (TGF-β1) regulates cellular proliferation, differentiation, migration, and survival. The human TGF-β1 transcript is inherently poorly translated, and translational activation has been documented in relation to several stimuli. In this paper, we have sought to identify in cis regulatory elements within the TGF-β1 5′Untranslated Region (5′UTR). In silico analysis predicted formation of stable secondary structure in a G/C-rich element between nucleotides +77 to +106, and demonstrated that this element is highly conserved across species. Circular dichroism spectroscopy confirmed the presence of secondary structure in this region. The proximal 5′UTR was inhibitory to translation in reporter gene experiments, and mutation of the secondary structure motif increased translational efficiency. Translational regulation of TGF-β1 mRNA is linked to altered binding of YB-1 protein to its 5′UTR. Immunoprecipitation-RT-qPCR demonstrated a high basal association of YB-1 with TGF-β1 mRNA. However, mutation of the secondary structure motif did not prevent interaction of YB-1 with the 5′UTR, suggesting that YB-1 binds to this region due to its G/C-rich composition, rather than a specific, sequence-dependent, binding site. These data identify a highly conserved element within the TGF-β1 5′UTR that forms stable secondary structure, and is responsible for the inherent low translation efficiency of this cytokine

Post-Transcriptional Regulation of Transforming Growth Factor Beta-1 by MicroRNA-744

Transforming Growth Factor Beta-1 (TGF-b1) is a pleiotropic cytokine that is of central importance in wound healing, inflammation, and in key pathological processes including cancer and progressive tissue fibrosis. TGF-b1 is posttranscriptionally regulated, but the underlying mechanisms remain incompletely defined. Previously, we have extensively delineated post-transcriptional regulation of TGF-b1 synthesis in the kidney, with evidence for relief of translational repression in proximal tubular cells in the context of diabetic nephropathy. In this study, we have investigated the role of the TGF-b1 39Untranslated Region (39UTR). Two different 39UTR lengths have been reported for TGF-b1, of 543 and 137 nucleotides. Absolute quantification showed that, while both UTR lengths were detectable in various human cell types and in a broad range of tissues, the short form predominated in the kidney and elsewhere. Expression of both forms was upregulated following auto-induction by TGF-b1, but the short:long UTR ratio remained constant. Incorporation of the short UTR into a luciferase reporter vector significantly reduced reporter protein synthesis without major effect on RNA amount, suggesting post-transcriptional inhibition. In silico approaches identified multiple binding sites for miR-744 located in the proximal TGF-b1 39UTR. A screen in RNA from human tissues showed widespread miR-744 expression. miR-744 transfection inhibited endogenous TGF-b1 synthesis, while direct targeting of TGF-b1 was shown in separate experiments, in which miR-744 decreased TGF-b1 39UTR reporter activity. This work identifies miR-744-directed post-transcriptional regulation of TGFb

Diagrammatic representation of the TGF-β₁ 5′UTR inhibitory element.

<p>The TGF-β₁ 5′UTR inhibitory element nucleotides +1 to 167 derived from the reference sequence: accession number NM_000660. The sequence in capitals identifies the G/C-rich stem loop secondary structure and the bold/underlined sequence indicates the previously identified putative YB-1 binding sites. The arrow indicates the start position of the numbering convention of Kim et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012283#pone.0012283-Kim1" target="_blank">[14]</a>.</p

Ensemble free energy per nucleotide <i>vs.</i> window centre.

<p>Ensemble free energy per nucleotide <i>vs.</i> window centre is plotted for 50 nucleotide sliding windows (solid line) of the TGF-β₁ 5′UTR. Mean ensemble free energy of 100 scrambled windows <i>vs.</i> window centre is plotted (dashed line).</p

A. Reporter gene analysis of the TGF-β₁ 5′UTR.

<p>Luminescence was measured by Dual Luciferase Reporter Assay Kit (Promega). Luciferase activity is expressed as normalised firefly luciferase activity relative to that of the pGL3-Control vector. Results shown represent mean (±SEM) of four experiments performed in triplicate. <b>B</b>. Quantification of luciferase mRNA by RT-qPCR according to standard protocol using SYBR® Green dye. Results shown represent mean (±SEM) of four experiments performed in triplicate. Statistical analysis was performed using an unpaired <i>t</i> test with Welch's correction (P<0.005***).</p

Oligonucleotide primer sequences, given in 5′-3′ orientation, for RT-qPCR, cDNA amplification and the anneal/extension of large overlapping primers to create the stem loop secondary structure and YB-1 binding site mutants.

<p>The altered residues in the mutants are denoted by capital letters.</p

<i>In silico</i>- and <i>In vitro-</i> analysis of nucleotides 77–106.

<p><b>A</b>. Centroid structure of the G/C-rich stem loop predicted between nucleotides 77–106 of the TGF-β₁ 5′UTR. <b>B</b>. Circular dichroism spectra (Δε) of the TGF-β₁ 5′UTR nucleotides 75–113 (7µM) in 10 mM Tris.HCl, pH 8, 100 mM KCl, 6 M urea. (i) solid line, initial spectrum at 5°C; (ii) dashed line, 95°C; (iii) dotted line, final spectrum at 5°C.</p

Comparison of translationally active and repressed mRNA.

<p>Northern blot of fractionated mRNA probed for <b>A</b>. TGF-β₁ and <b>B</b>. β-actin as described in the materials and methods. <b>C</b>. Graphical representation of polysome distribution of β-actin <b>(shaded area)</b> and TGF-β₁<b>(solid line)</b>, expressed as a percentage of total mRNA detected on a given blot in each fraction. <b>D</b>. Immunoprecipitation of YB-1/mRNA complexes and RT-qPCR for β-actin and TGF-β₁. The results are representative of four individual experiments and where appropriate represent mean (±SEM) performed in triplicate. Statistical analysis was performed using an unpaired <i>t</i> test with Welch's correction (P<0.005***).</p

Reporter gene analysis of TGF-β₁ 5′UTR mutations.

<p><b>A</b>. Schematic diagram of the TGF-β₁ 5′UTR endogenous and mutant 167-nucleotide fragments as described in the methods. Open boxes indicate the stem loop secondary structure, filled boxes indicated the position of the adenine residue substitutions. <b>B</b>. Luminescence was measured by Dual Luciferase Reporter Assay Kit (Promega). Luciferase activity is expressed as normalised firefly luciferase activity relative to that of the pGL3-Control vector. Results shown represent mean (±SEM) of four experiments performed in triplicate. Statistical analysis was performed using an unpaired <i>t</i> test with Welch's correction (P<0.05**, P<0.005***).</p