Summary RNA transcripts are subject to post-transcriptional gene regulation involving hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) expressed in a cell-type dependent fashion. We developed a cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs. The crosslinked sites are revealed by thymidine to cytidine transitions in the cDNAs prepared from immunopurified RNPs of 4-thiouridine-treated cells. We determined the binding sites and regulatory consequences for several intensely studied RBPs and miRNPs, including PUM2, QKI, IGF2BP1-3, AGO/EIF2C1-4 and TNRC6A-C. Our study revealed that these factors bind thousands of sites containing defined sequence motifs and have distinct preferences for exonic versus intronic or coding versus untranslated transcript regions. The precise mapping of binding sites across the transcriptome will be critical to the interpretation of the rapidly emerging data on genetic variation between individuals and how these variations contribute to complex genetic diseases.

Defects in insulin signalling are among the most common and earliest defects that predispose an individual to the development of type 2 diabetes. MicroRNAs have been identified as a new class of regulatory molecules that influence many biological functions, including metabolism. However, the direct regulation of insulin sensitivity by microRNAs in vivo has not been demonstrated. Here we show that the expression of microRNAs 103 and 107 (miR-103/107) is upregulated in obese mice. Silencing of miR-103/107 leads to improved glucose homeostasis and insulin sensitivity. In contrast, gain of miR-103/107 function in either liver or fat is sufficient to induce impaired glucose homeostasis. We identify caveolin-1, a critical regulator of the insulin receptor, as a direct target gene of miR-103/107. We demonstrate that caveolin-1 is upregulated upon miR-103/107 inactivation in adipocytes and that this is concomitant with stabilization of the insulin receptor, enhanced insulin signalling, decreased adipocyte size and enhanced insulin-stimulated glucose uptake. These findings demonstrate the central importance of miR-103/107 to insulin sensitivity and identify a new target for the treatment of type 2 diabetes and obesity.

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...