Mung Bean nuclease mapping of RNAs 3' end

A method is described that allows an accurate mapping of 3' ends of RNAs. In this method a labeled DNA probe, containing the presumed 3' end of the RNA under analysis is allowed to anneals to the RNA itself. Mung-bean nuclease is then used to digest single strands of both RNA and DNA. Electrophoretic fractionation of "protected" undigested, labeled DNA is than performed using a sequence reaction of a known DNA as length marker. This procedure was applied to the analysis of both a polyA RNA (Interleukin 10 mRNA) and non polyA RNAs (sea urchin 18S and 26S rRNAs). This method might be potentially relevant for the evaluation of the role of posttrascriptional control of IL-10 in the pathogenesis of the immune and inflammatory mediated diseases associated to ageing. This might allow to develop new strategies to approach to the diagnosis and therapy of age related diseases

Thymus-specific deletion of insulin induces autoimmune diabetes

Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance towards pancreatic β-cells. To further explore the function of this ectopic insulin expression, we knocked out the mouse Ins2 gene specifically in the Aire-expressing medullary thymic epithelial cells (mTECs), without affecting its expression in the β-cells. When further crossed to the Ins1 knockout background, both male and female pups (designated as ID-TEC mice for insulin-deleted mTEC) developed diabetes spontaneously around 3 weeks after birth. β-cell-specific autoimmune destruction was observed, as well as islet-specific T cell infiltration. The presence of insulin-specific effector T cells was shown using ELISPOT assays and adoptive T cell transfer experiments. Results from thymus transplantation experiments proved further that depletion of Ins2 expression in mTECs was sufficient to break central tolerance and induce anti-insulin autoimmunity. Our observations may explain the rare cases of type 1 diabetes onset in very young children carrying diabetes-resistant HLA class II alleles. ID-TEC mice could serve as a new model for studying this pathology

Long noncoding RNAs are dynamically regulated during β-cell mass expansion in mouse pregnancy and control β-cell proliferation <i>in vitro</i>

<div><p>Pregnancy is associated with increased β-cell proliferation driven by prolactin. Long noncoding RNAs (lncRNA) are the most abundant RNA species in the mammalian genome, yet, their functional importance is mainly elusive. <b>Aims/hypothesis</b>: This study tests the hypothesis that lncRNAs regulate β-cell proliferation in response to prolactin in the context of β-cell mass compensation in pregnancy. <b>Methods</b>: The expression profile of lncRNAs in mouse islets at day 14.5 of pregnancy was explored by a bioinformatics approach, further confirmed by quantitative PCR at different days of pregnancy, and islet specificity was evaluated by comparing expression in islets versus other tissues. In order to establish the role of the candidate lncRNAs we studied cell proliferation in mouse islets and the MIN6 β-cell line by EdU incorporation and cell count. <b>Results</b>: We found that a group of lncRNAs is differentially regulated in mouse islets at 14.5 days of pregnancy. At different stages of pregnancy, these lncRNAs are dynamically expressed, and expression is prolactin dependent in mouse islets and MIN6 cells. One of those lncRNAs, Gm16308 (Lnc03), is dynamically regulated during pregnancy, prolactin-dependent and islet-enriched. Silencing Lnc03 in primary β-cells and MIN6 cells inhibits, whereas over-expression stimulates, proliferation even in the absence of prolactin, demonstrating that Lnc03 regulates β-cell growth. <b>Conclusions/interpretation</b>: During pregnancy mouse islet proliferation is correlated with dynamic changes of lncRNA expression. In particular, Lnc03 regulates mouse β-cell proliferation and may be a crucial component of β-cell proliferation in β-cell mass adaptation in both health and disease.</p></div

Analysis of Lnc03 expression in MIN6 cells.

<p>a) MIN6 cells were treated with 200 or 500ng/ml Prl (200 Prl, 500 Prl) for 24h (grey bars) and 48h (black bars), non-treated cells were used as control (C, white bar). Total RNA was used to perform RT-qPCR. Data are presented as fold change versus C, mean±SEM, n = 3. b) Lnc03 expression was evaluated in a time course experiments in MIN6 cell treated with 500ng/ml Prl (black bars). 0h (white bar) represents non-treated control cells. Data are presented as fold change versus 0h, mean±SEM, n = 4. c) MIN6 cells were treated with 100μm Stat5 inhibitor alone (Sta5in) or together with 500ng/ml Prl (Sta5in+Prl) or Prl alone for 24h. Control cells were not treated (C white bar). Data are presented as fold change versus C, mean±SEM, n = 4–8.</p

Expression of candidate lncRNAs in isolated mouse islets treated with Prl.

<p>Isolated mouse islets were treated with 200ng/ml or 500ng/ml (200 Prl, 500 Prl) for 24h or 48h; non-treated islets were used as control (C). Total RNA was used to perform RT-qPCR. Data are presented as fold change versus C, mean±SEM, n = 3. Statistically significant differences were determined using One-way ANOVA with Bonferroni post hoc test. *p< 0.05; **p < 0.01; ***p < 0.001.</p

Expression of candidate lncRNAs in 8 different mouse tissues.

<p>Total RNA from control animals was used to perform RT-qPCR of Lnc01-Lnc06. The results were normalized to Hprt (hypoxanthine guanine phosphoribosyl transferase), and Gapdh (glyceraldehyde 3-phosphate dehydrogenase) expressed as fold change versus islets. Data are shown as mean±SEM, n = 4–8.</p

Identification of differentially expressed lncRNAs in mouse islets during pregnancy.

<p>Differentially expressed non-coding RNAs at gestational day 14.5 identified by RNA-seq analysis were filtered to remove rRNAs, snoRNAs, and miRNAs. The log2-transformed fold change of remaining ncRNAs is compared with microarray analysis. Five potential lncRNAs were selected. An additional lncRNA Cuff.9036 identified by Cufflinks (GeneBank accession MF497421), as a novel transcript only present in our annotation, was also selected for further study (table in Fig 1).</p