MicroRNAs are small noncoding RNAs that recognize and bind to partially complementary sites in the 3' untranslated regions of target genes in animals and, by unknown mechanisms, regulate protein production of the target transcript. Different combinations of microRNAs are expressed in different cell types and may coordinately regulate cell-specific target genes. Here, we present PicTar, a computational method for identifying common targets of microRNAs. Statistical tests using genome-wide alignments of eight vertebrate genomes, PicTar's ability to specifically recover published microRNA targets, and experimental validation of seven predicted targets suggest that PicTar has an excellent success rate in predicting targets for single microRNAs and for combinations of microRNAs. We find that vertebrate microRNAs target, on average, roughly 200 transcripts each. Furthermore, our results suggest widespread coordinate control executed by microRNAs. In particular, we experimentally validate common regulation of Mtpn by miR-375, miR-124 and let-7b and thus provide evidence for coordinate microRNA control in mammals.

MicroRNAs (miRNAs) constitute a growing class of non-coding RNAs that are thought to regulate gene expression by translational repression. Several miRNAs in animals exhibit tissue-specific or developmental-stage-specific expression, indicating that they could play important roles in many biological processes. To study the role of miRNAs in pancreatic endocrine cells we cloned and identified a novel, evolutionarily conserved and islet-specific miRNA (miR-375). Here we show that overexpression of miR-375 suppressed glucose-induced insulin secretion, and conversely, inhibition of endogenous miR-375 function enhanced insulin secretion. The mechanism by which secretion is modified by miR-375 is independent of changes in glucose metabolism or intracellular Ca2+-signalling but correlated with a direct effect on insulin exocytosis. Myotrophin (Mtpn) was predicted to be and validated as a target of miR-375. Inhibition of Mtpn by small interfering (si)RNA mimicked the effects of miR-375 on glucose-stimulated insulin secretion and exocytosis. Thus, miR-375 is a regulator of insulin secretion and may thereby constitute a novel pharmacological target for the treatment of diabetes.

In stratified epithelial tissues, homeostasis relies on the self-renewing capacity of stem cells located within the innermost basal layer. As basal cells become suprabasal, they lose proliferative potential and embark on a terminal differentiation programme. Here, we show that microRNA-203 is induced in the skin concomitantly with stratification and differentiation. By altering miR-203's spatiotemporal expression in vivo, we show that miR-203 promotes epidermal differentiation by restricting proliferative potential and inducing cell-cycle exit. We identify p63 as one of the conserved targets of miR-203 across vertebrates. Notably, p63 is an essential regulator of stem-cell maintenance in stratified epithelial tissues. We show that miR-203 directly represses the expression of p63: it fails to switch off suprabasally when either Dicer1 or miR-203 is absent and it becomes repressed basally when miR-203 is prematurely expressed. Our findings suggest that miR-203 defines a molecular boundary between proliferative basal progenitors and terminally differentiating suprabasal cells, ensuring proper identity of neighbouring layers.

Altered growth and development of the endocrine pancreas is a frequent cause of the hyperglycemia associated with diabetes. Here we show that microRNA-375 (miR-375), which is highly expressed in pancreatic islets, is required for normal glucose homeostasis. Mice lacking miR-375 (375KO) are hyperglycemic, exhibit increased total pancreatic ␣-cell numbers, fasting and fed plasma glucagon levels, and increased gluconeogenesis and hepatic glucose output. Furthermore, pancreatic ␤-cell mass is decreased in 375KO mice as a result of impaired proliferation. In contrast, pancreatic islets of obese mice (ob/ob), a model of increased ␤-cell mass, exhibit increased expression of miR-375. Genetic deletion of miR-375 from these animals (375/ob) profoundly diminished the proliferative capacity of the endocrine pancreas and resulted in a severely diabetic state. Bioinformatic analysis of transcript data from 375KO islets revealed that miR-375 regulates a cluster of genes controlling cellular growth and proliferation. These data provide evidence that miR-375 is essential for normal glucose homeostasis, ␣-and ␤-cell turnover, and adaptive ␤-cell expansion in response to increasing insulin demand in insulin resistance.diabetes ͉ glucagon ͉ microRNA ͉ islet ͉ proliferation T he maintenance of ␤-cell mass during development and throughout life is a highly regulated process responsible for normal glucose homeostasis. Defects in the development of pancreatic islets lead to changes in islet composition, and they often result in the hyperglycemia that characterizes the diabetic state (1, 2). The dynamic adaptation of ␤-cell mass in adult life is influenced by various metabolic stresses, which control the balance between proliferation and apoptosis. These processes, known to be regulated at the transcriptional level, contribute to the development and maintenance of many tissues, including the pancreatic islet (3, 4). Recent studies have shown that microRNAs (miRNAs), which regulate gene expression at a posttranscriptional level, are powerful regulators of growth, differentiation, and organ function (5-7). For instance, mutant mice in which miRNAs are collectively silenced during endocrine pancreas development exhibit defects in all pancreatic lineages, including a dramatic reduction of insulin-producing ␤ cells (8). It is estimated that Ϸ30% of all protein coding genes are miRNA targets. Combining target prediction with experimental analysis of miRNA expression and production of loss of function mutants is beginning to improve our understanding of the roles that miRNAs play in normal and disease states (7-12). We have previously reported that miR-375, the highest expressed miRNA in pancreatic islets of humans and mice, regulates insulin secretion in isolated pancreatic ␤ cells (13). In this study we have investigated the effect of genetic ablation of miR-375 on pancreatic islet development and function and in the etiology of type 2 diabetes. Results Development of Hyperglycemia in miR-375-Null Mice.To elucidate the role of miR-375 in th...