The use of alternative polyadenylation sites renders integrin β1 (Itgb1) mRNA isoforms with differential stability during mammary gland development

Integrins are heterodimeric cell-surface adhesion receptors that play a critical role in tissue development. Characterization of the full-length mRNA encoding the β1 subunit (Itgb1) revealed an alternative functional cleavage and polyadenylation site that yields a new Itgb1 mRNA isoform 578 bp shorter than that previously reported. Using a variety of experimental and bioinformatic approaches, we found that the two Itgb1 isoforms are expressed at different levels in a variety of mouse tissues, including the mammary gland, where they are differentially regulated at successive developmental stages. The longer mRNA species is prevelant during lactation, whereas the shorter is induced after weaning. In 3D cultures, where expression of integrin β1 protein is required for normal formation of acini, experimental blockade of the longer isoform induced enhanced expression of the shorter species which allowed normal morphological mammary differentiation. The short isoform lacks AU-rich motifs and miRNA target sequences that are potentially implicated in the regulation of mRNA stability and translation efficiency. We further determined that the AU-binding protein HuR appears to selectively stabilize the longer isoform in the mammary gland. In summary, the results of the present study identify a new regulatory instance involved in the fine-tuning of Itgb1 expression during mammary gland development and function.Centro de Investigaciones Inmunológicas Básicas y Aplicada

Mammary differentiation induces expression of Tristetraprolin, a tumor suppressor AU-rich mRNA-binding protein

Tristetraprolin (TTP) is a RNA-binding protein that inhibits the expression of pro-inflammatory cytokines and invasiveness-associated genes. TTP levels are decreased in many different cancer types and it has been proposed that this protein could be used as a prognostic factor in breast cancer. Here, using publicly available DNA microarray datasets, “serial analysis of gene expression” libraries and qRT-PCR analysis, we determined that TTP mRNA is present in normal breast cells and its levels are significantly decreased in all breast cancer subtypes. In addition, by immunostaining, we found that TTP expression is higher in normal breast tissue and benign lesions than in infiltrating carcinomas. Among these, lower grade tumors showed increased TTP expression compared to higher grade cancers. Therefore, these data indicate that TTP protein levels would provide a better negative correlation with breast cancer invasiveness than TTP transcript levels. In mice, we found that TTP mRNA and protein expression is also diminished in mammary tumors. Interestingly, a strong positive association of TTP expression and mammary differentiation was identified in normal and tumor cells. In fact, TTP expression is highly increased during lactation, showing good correlation with various mammary differentiation factors. TTP expression was also induced in mammary HC11 cells treated with lactogenic hormones, mainly by prolactin, through Stat5A activation. The effect of this hormone was highly dependent on mammary differentiation status, as prolactin was unable to elicit a similar response in proliferating or neoplastic mammary cells. In summary, these studies show that TTP expression is strongly linked to the mammary differentiation program in human and mice, suggesting that this protein might play specific and relevant roles in the normal physiology of the gland.Facultad de Ciencias MédicasCentro de Investigaciones Inmunológicas Básicas y Aplicada

BUHO: A MATLAB Script for the Study of Stress Granules and Processing Bodies by High-Throughput Image Analysis

<div><p>The spontaneous and reversible formation of <em>foci</em> and filaments that contain proteins involved in different metabolic processes is common in both the nucleus and the cytoplasm. Stress granules (SGs) and processing bodies (PBs) belong to a novel family of cellular structures collectively known as mRNA silencing <em>foci</em> that harbour repressed mRNAs and their associated proteins. SGs and PBs are highly dynamic and they form upon stress and dissolve thus releasing the repressed mRNAs according to changes in cell physiology. In addition, aggregates containing abnormal proteins are frequent in neurodegenerative disorders. In spite of the growing relevance of these supramolecular aggregates to diverse cellular functions a reliable automated tool for their systematic analysis is lacking. Here we report a MATLAB Script termed BUHO for the high-throughput image analysis of cellular <em>foci</em>. We used BUHO to assess the number, size and distribution of distinct objects with minimal deviation from manually obtained parameters. BUHO successfully addressed the induction of both SGs and PBs in mammalian and insect cells exposed to different stress stimuli. We also used BUHO to assess the dynamics of specific mRNA-silencing <em>foci</em> termed Smaug 1 <em>foci</em> (S-<em>foci</em>) in primary neurons upon synaptic stimulation. Finally, we used BUHO to analyze the role of candidate genes on SG formation in an RNAi-based experiment. We found that FAK56D, GCN2 and PP1 govern SG formation. The role of PP1 is conserved in mammalian cells as judged by the effect of the PP1 inhibitor salubrinal, and involves dephosphorylation of the translation factor eIF2α. All these experiments were analyzed manually and by BUHO and the results differed in less than 5% of the average value. The automated analysis by this user-friendly method will allow high-throughput image processing in short times by providing a robust, flexible and reliable alternative to the laborious and sometimes unfeasible visual scrutiny.</p> </div

The use of alternative polyadenylation sites renders integrin beta1 (Itgb1) mRNA isoforms with differential stability during mammary gland development

Integrins are heterodimeric cell-surface adhesion receptors that play a critical role in tissue development. Characterization of the full-length mRNA encoding the beta1 subunit (Itgb1) revealed an alternative functional cleavage and polyadenylation site that yields a new Itgb1 mRNA isoform 578 bp shorter than that previously reported. Using a variety of experimental and bioinformatic approaches, we found that the two Itgb1 isoforms are expressed at different levels in a variety of mouse tissues, including the mammary gland, where they are differentially regulated at successive developmental stages. The longer mRNA species is prevelant during lactation, whereas the shorter is induced after weaning. In 3D cultures, where expression of integrin beta1 protein is required for normal formation of acini, experimental blockade of the longer isoform induced enhanced expression of the shorter species which allowed normal morphological mammary differentiation. The short isoform lacks AU-rich motifs and miRNA target sequences that are potentially implicated in the regulation of mRNA stability and translation efficiency. We further determined that the AU-binding protein HuR appears to selectively stabilize the longer isoform in the mammary gland. In summary, the results of the present study identify a new regulatory instance involved in the fine-tuning of Itgb1 expression during mammary gland development and function

Analysis of PB induction by BUHO.

<p>Fly (A) or mammalian cells (B) were exposed to oxidative stress as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051495#s4" target="_blank">Materials and Methods</a> and PBs were immunostained. PB size was evaluated using BUHO in 55 resting and 68 stressed S2R+ cells, and in 15 resting and 23 stressed U2OS cells, and by manual counting in 10 stressed or 10 resting S2R+ representative cells and 9 stressed and 9 resting representative U2OS cells. 63×, 1024×1024 (A) or 40×, 2048×2048 (B) confocal images were used. Size bar, 10 µm.</p

Summary of manual and BUHO analysis for the identification of SG regulators.

<p>S2R+ cells were treated with the indicated dsRNA and exposed to arsenite. SG formation was analyzed in 7 images (test set) with an average of 300 cells for each treatment. Manual values and BUHO generated results are indicated.</p

Redundant SG recognition by distinct prototypes.

<p>The percentage of SGs recognized by each prototype, or simultaneously by pairs of prototypes in the 6 representative micrographs of the training set, with a total of 282 granules is indicated. ST was 0.89 for prototype I; 0.88 for prototype II; 0.8 for prototype III; 0.88 for prototype IV; 0.86 for prototype V; 0.86 for prototype VI; 0.92 for prototype VII; and 0.85 for prototype VIII. Redundancy allowed a single SG to be recognized by multiple prototypes.</p

PP1α governs SG disassembly.

<p>A, <i>Drosophila</i> S2R+ cells were exposed to the indicated dsRNA and the effect on SG formation was evaluated. Triplicate wells of the indicated RNAi treatments were analyzed both manually and by BUHO. 204 control wells were analyzed by BUHO, and a subset of 6 wells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051495#pone-0051495-t003" target="_blank">Table 3</a>) was analyzed manually. The score is the ratio of the percentage cells with SGs in each well relative to the average percentage of cells with SGs in the control wells in the same plate. GCN2 knockdown impaired SG formation, and the KD of FAK56D or PP1α-96A facilitated their assembly. Scores determined manually differ in less than 7% from those calculated by BUHO. B and C, Mammalian U2OS cells were exposed to ER-stress in the presence (Sal) or absence (none) of salubrinal, as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051495#s4" target="_blank">Materials and Methods</a>. B, The number of SG-positive cells at the indicated time points was evaluated manually (M) or by BUHO (B) in 63×, 1024×1024 images. Error bars, standard deviation. C, Phosphorylation of eIF2α relative to basal conditions was evaluated three hours after stress induction in the presence or absence of salubrinal, as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051495#s4" target="_blank">Materials and Methods</a>. Error bars, standard deviation.</p

S-<i>foci</i> dissolution upon synaptic stimulation.

<p>Cultured rat neurons were stimulated with NMDA and S-<i>foci</i> and synapses were identified with specific antibodies as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051495#s4" target="_blank">Materials and Methods</a>. The S-<i>foci</i> size and the number of synapses containing S-<i>foci</i> in their surroundings at a distance lower than 0.5 µm were evaluated. S-<i>foci</i> smaller than 0.2 µm² were not included. Six micrographs (63×, 1024×1024 pixels), containing 4180 synapses were analyzed using BUHO, and a subset of 200 representative synapses were analyzed manually. Values relative to basal conditions are plotted. Error bars indicate standard deviation. Size bar, 2 µm.</p