15 research outputs found
A bus as a public space. Multiculturalism and Urban Ethnography in Milan
<p></p><p>Abstract The paper draws on a research and teaching project carried out with an international class of students in Urban Ethnography in the MSC in Urban Planning and Policy at Politecnico di Milano (Italy). A particular focus of the project was on exploring the role played by public spaces in supporting the coexistence of a multitude of strangers in the city through the continuous negotiation of diversity and difference. In the field work, spatial and social dynamics occurring in a particular and ‘compressed’ public space - the 90-91 trolley-bus circle-line in Milan - are explored and discussed as a space of confrontation in a perspective of daily multiculturalism.</p><p></p
Let-7b/c Enhance the Stability of a Tissue-Specific mRNA during Mammalian Organogenesis as Part of a Feedback Loop Involving KSRP
<div><p>Gene silencing mediated by either microRNAs (miRNAs) or Adenylate/uridylate-rich elements Mediated mRNA Degradation (AMD) is a powerful way to post-transcriptionally modulate gene expression. We and others have reported that the RNA–binding protein KSRP favors the biogenesis of select miRNAs (including let-7 family) and activates AMD promoting the decay of inherently labile mRNAs. Different layers of interplay between miRNA– and AMD–mediated gene silencing have been proposed in cultured cells, but the relationship between the two pathways in living organisms is still elusive. We conditionally deleted <em>Dicer</em> in mouse pituitary from embryonic day (E) 9.5 through Cre-mediated recombination. <em>In situ</em> hybridization, immunohistochemistry, and quantitative reverse transcriptase–PCR revealed that Dicer is essential for pituitary morphogenesis and correct expression of hormones. Strikingly, αGSU (alpha glycoprotein subunit, common to three pituitary hormones) was absent in <em>Dicer</em>-deleted pituitaries. αGSU mRNA is unstable and its half-life increases during pituitary development. A transcriptome-wide analysis of microdissected E12.5 pituitaries revealed a significant increment of KSRP expression in conditional <em>Dicer</em>-deleted mice. We found that KSRP directly binds to αGSU mRNA, promoting its rapid decay; and, during pituitary development, αGSU expression displays an inverse temporal relationship to KSRP. Further, let-7b/c downregulated KSRP expression, promoting the degradation of its mRNA by directly binding to the 3′UTR. Therefore, we propose a model in which let-7b/c and KSRP operate within a negative feedback loop. Starting from E12.5, KSRP induces the maturation of let-7b/c that, in turn, post-transcriptionally downregulates the expression of KSRP itself. This event leads to stabilization of αGSU mRNA, which ultimately enhances the steady-state expression levels. We have identified a post-transcriptional regulatory network active during mouse pituitary development in which the expression of the hormone αGSU is increased by let7b/c through downregulation of KSRP. Our study unveils a functional crosstalk between miRNA– and AMD–dependent gene regulation during mammalian organogenesis events.</p> </div
KSRP is required for αGSU mRNA degradation.
<p>(A) Anti-KSRP antibody immunoprecipitates αGSU mRNA in αT3-1 cell extracts. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) UV-crosslink assay to analyze the interaction of the recombinant KSRP (40–200 nM) as well as the indicated KSRP deletion mutants (150 nM) with radiolabeled αGSU 3′UTR or pitx2 3′UTR stable region (E3) as a negative control <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002823#pgen.1002823-Briata2" target="_blank">[38]</a>. (C) Luciferase reporter plasmid bearing either wt or Δ-ARE αGSU 3′UTR sequences were cotransfected into HeLa cells with either a vector overexpressing KSRP or siRNA knocking it down. Quantitative RT-PCR analysis of luciferase transcript was normalized using Renilla mRNA. siCTL, control siRNA (D) Quantitative RT-PCR analysis of αGSU transcript in either sh-KSRP or empty pSUPER vector stably transfected into αT3-1 cells. Total RNA was isolated at the indicated times after addition of actinomycin D. The data were normalized by β2-MG mRNA. (E) <i>In vitro</i> RNA degradation assays were performed by incubating S100s from mock αT3-1, αT3-1–shKSRP, or -overexpressing KSRP cells with either wt or Δ-ARE mutant αGSU 3′UTR internally <sup>32</sup>P-labeled and capped RNA substrates. The decay was monitored at the indicated times. (F) RT-PCR analysis of both αGSU and β2-MG transcripts from either mock αT3-1 or αT3-1–shKSRP cells. Student's t-test: *P<0.05. All data are presented as mean and s.d. (n = 4).</p
A model for the miRNA–dependent control of αGSU transcript stability during pituitary development.
<p>A model for the miRNA–dependent control of αGSU transcript stability during pituitary development.</p
Dicer is required for cell-type-specific pituitary hormone expression.
<p>Protein expression of TSHβ, GH, POMC (expression was detected using anti-ACTH immunoglobulin G), LHβ, and αGSU in control or <i>Dicer</i>-deleted pituitaries at E17.5 by immunohistochemistry; a representative sagittal section of pituitary gland is shown.</p
Dicer regulates αGSU and KSRP expression during pituitary development.
<p>(A) <i>In situ</i> hybridization (upper panels) and immunohistochemical analyses of αGSU (middle panels) and KSRP (lower panels) in control or <i>Dicer</i>-deleted pituitaries at E12.5; a representative sagittal section of pituitary gland is shown. (B) Reverse transcription (RT) followed by quantitative PCR to analyze αGSU (upper panel) and KSRP (lower panel) mRNA expression in control or <i>Dicer</i>-deleted pituitaries at E12.5, E15.5, and E17.5. The data were normalized by β2-MG mRNA. a.u., arbitrary units compared to the value of Dicer<sup>wt/flox</sup> at E12.5. (C) immunoblot analysis of αGSU, KSRP and β-tubulin from several pituitary cell lines as indicated (GC-somatotrope, ATT20-corticotrope, αT3-1-gonadotrope, MMQ-lactotrope, and TαT1-thyrotrope). (D) Quantitative RT-PCR analysis of αGSU transcript in αT3-1 and TαT-1 cells. Total RNA was isolated at the indicated times after addition of actinomycin D. The data were normalized by β2-MG mRNA. All data are presented as mean and s.d. (n = 4).</p
Let-7b and let-7c directly downregulate KSRP mRNA during pituitary development.
<p>(A) Ontogeny of let-7c expression in pituitary development by quantitative RT-PCR. The data were normalized by U6 RNA. (B) Immunoblot analysis of KSRP and β-tubulin in NIH-3T3 cells singularly transfected with the indicated miRNA mimics. (C) RNA duplex expected to result from base pairing of KSRP mRNA with let-7c. (D) Relative luciferase activity of reporter constructs containing four either wt or mutant let-7b/c binding sites from KSRP 3′UTR sequence in 293T cells overexpressing pri-let-7c-1. The data were normalized using Renilla activity. (E) Ontogeny of pri-let-7b/c-2, and (F) Lin28A mRNA expression in pituitary development by quantitative RT-PCR. The data were normalized by β2-MG mRNA. Student's t-test: **P<0.01. All data are presented as mean and s.d. (n = 4).</p
PI3K-AKT signaling stabilizes a set of KSRP-interacting mRNAs and increases their expression
<p><b>Copyright information:</b></p><p>Taken from "Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling"</p><p>http://www.biomedcentral.com/1471-2199/8/28</p><p>BMC Molecular Biology 2007;8():28-28.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1858702.</p><p></p> (A) Either mock-αT3-1 or αT3-1-myrAKT1 cells were lysed and total extracts were immunoprecipitated (Ip) with either anti-AKT antibody or control IgG (cIgG). Pellets were incubated (20 min at 30°C) with histone 2B (H2B) in kinase buffer in the presence of γ[P]ATP under gentle shaking. Labeled proteins were separated by SDS-PAGE and detected by autoradiography. (B) Expression of KSRP-interacting mRNAs and β2-MG (control transcript), monitored by RT-PCR, in either mock-αT3-1 or αT3-1-myrAKT1 cells. (C) Semi quantitative RT-PCR analysis of both KSRP-interacting mRNAs and β2-MG (control transcript) in either mock-αT3-1 (red lines) or αT3-1-myrAKT1 (blue lines). Total RNA was isolated at the indicated times after addition of Actinomycin D. The amount of each transcript was quantitated by densitometry and plotted using a linear regression program. The values shown are averages (± SEM) of three independent experiments performed in duplicates. A quantitation of the transcripts' t(1/2) is presented in Additional file
KSRP associates with AUF1p45 and hnRNPA1 in cytoplasmic extracts of aT3-1 cells
<p><b>Copyright information:</b></p><p>Taken from "Identification of a set of KSRP target transcripts upregulated by PI3K-AKT signaling"</p><p>http://www.biomedcentral.com/1471-2199/8/28</p><p>BMC Molecular Biology 2007;8():28-28.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1858702.</p><p></p> (A) S100 extracts from αT3-1 cells were subjected to gel filtration chromatography on a Superose 6 column. Aliquots of the eluted fractions were analyzed by Western blotting using the indicated antibodies. (B) RNase A-treated S100 extracts from αT3-1 cells were immunoprecipitated with preimmune (lane 2) or anti-KSRP (lane 3) sera and analyzed by immunoblotting with either anti-AUF1 (top) or anti-HnRNPA1 (bottom) antibodies. The arrows mark the position of either AUF1p45 or hnRNPA1, while the asterisk marks a anti-AUF1 cross-reacting band. (C) GST-pulldown of either endogenous AUF1p45 (top) or endogenous hnRNPA1 (bottom) from S100 extracts of αT3-1 cells using either control GST or GST-KSRP. Proteins were analyzed by immunoblotting using the indicated antibodies. The arrows mark the position of either AUF1p45 or hnRNPA1
mRNA Encoding β-Catenin Is Labile and Is Stabilized by LiCl and Wnt-3A
<div><p>(A) Expression of β-catenin and β2-MG (control transcript), monitored by RT-PCR, in control and in LiCl-treated (20 mM for 6 h) αT3-1 cells.</p>
<p>(B) Quantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in αT3-1 cells. Total RNA was isolated at the indicated times after addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate.</p>
<p>(C) Quantitative RT-PCR analysis of both CAT and CAT–β-catenin transcripts in either CAT or CAT–β-catenin transiently transfected αT3-1 cells. Total RNA was isolated at the indicated times after addition of actinomycin D. The values shown are averages (±SEM) of three independent experiments performed in duplicate.</p>
<p>(D) Semiquantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in either control or LiCl-treated αT3-1 cells. Total RNA was isolated at the indicated times after the addition of actinomycin D. The amount of each transcript was quantitated by densitometry and plotted using a linear regression program. The values shown are averages (±SEM) of three independent experiments performed in duplicate.</p>
<p>(E) Expression of β-catenin and β2-MG, monitored by RT-PCR, in either control-treated αT3-1, or Wnt-3A–treated (10 ng/ml, 6 h) αT3-1 cells.</p>
<p>(F) Semiquantitative RT-PCR analysis of both β-catenin and β2-MG transcripts in either control or Wnt-3A–treated αT3-1 cells. Total RNA was isolated at the indicated times after the addition of actinomycin D. The amount of each transcript was quantitated and represented as described in (D).</p></div