MicroRNAs: Genomics, Biogenesis, Mechanism, and Function

The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

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Initial sequencing and analysis of the human genome.

抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

http://bartellab.wi.mit.edu/publication_reprints/Bartel_Cell09.pdf

MicroRNAs: Target Recognition and Regulatory Functions

A procedure for extracting plasmid DNA from bacterial cells is described. The method is simple enough to permit the analysis by gel electrophoresis of 100 or more clones per day yet yields plasmid DNA which is pure enough to be digestible by restriction enzymes. The principle of the method is selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double-stranded. Adequate pH control is accomplished without using a pH meter. Upon neutralization, chromosomal DNA renatures to form an insoluble clot, leaving plasmid DNA in the supernatant. Large and small plasmid DNAs have been extracted by this method.

A rapid alkaline extraction procedure for screening recombinant plasmid DNA

MicroRNAs (miRNAs) are noncoding RNAs that suppress translation of specific mRNAs. The miRNA machinery interacts with fragile X mental retardation protein (FMRP), which functions as translational repressor. We show that miR-125b and miR-132, as well as several other miRNAs, are associated with FMRP in mouse brain. miR-125b and miR-132 had largely opposing effects on dendritic spine morphology and synaptic physiology in hippocampal neurons. FMRP knockdown ameliorates the effect of miRNA overexpression on spine morphology. We identified NMDA receptor subunit NR2A as a target of miR-125b and show that NR2A mRNA is specifically associated with FMRP in brain. In hippocampal neurons, NR2A expression is negatively regulated through its 3' UTR by FMRP, miR-125b, and Argonaute 1. Regulation of NR2A 3'UTR by FMRP depends in part on miR-125b. Because NMDA receptor subunit composition profoundly affects synaptic plasticity, these observations have implications for the pathophysiology of fragile X syndrome, in which plasticity is altered.

Regulation of Synaptic Structure and Function by FMRP-Associated MicroRNAs miR-125b and miR-132

Introduction: Iron and copper are absorbed in their reduced forms (ferrous and cuprous, respectively). Duodenal cytochrome b (Dcytb) functions as an intestinal ferric reductase; however, the nature of the putative intestinal cupric reductase remains unclear. We have shown previously that cells over-expressing Dcytb show not only enhanced ferric reductase activity but also display significant cupric reductase activity, suggesting that Dcytb may function as a dual metallo-reductase in intestinal enterocytes (Wyman et al FEBS Lett 2008, 582:1901-6). The aims of this study were to investigate whether intestinal Dcytb expression was regulated by copper, and whether Dcytb might play a role in cellular copper uptake. Methods and Materials: Human intestinal epithelial cells (Caco-2 and HuTu-80) were exposed to either the copper chelator TETA (0.5 mM) or copper (50 µM) for 24 h. Cells were harvested for RNA and expression of Dcytb and the copper transporter Ctr1 determined by qPCR. To determine whether Dcytb plays a role in copper uptake we employed a MDCK cell line which over-expresses Dcytb under the control of a tetracycline-repressible promoter (TET-Off MDCK, Clontech; Wyman et al 2008). Copper uptake was measured by ICP-OES. Results: In Caco-2 cells, Ctr1 expression was significantly increased by TETA treatment (p< 0.01) and significantly decreased by exposure to high copper medium (p<0.05). Basal Dcytb expression in Caco-2 cells was below the detection threshold in our studies so we used HuTu-80 cells as an alternative intestinal model. Dcytb expression was significantly increased in TETA-treated cells (p<0.01). Copper treatment did not significantly alter Dcytb mRNA compared with untreated controls. Copper uptake in Dcytb-overexpressing MDCK cells was significantly increased compared with untransfected control cells (+6.7-fold; p<0.01). Treatment with doxycycline (20ng/ml) significantly reduced copper uptake to levels seen in untransfected cells (p<0.01). Conclusion: These data demonstrate that Dcytb is a copper-regulated gene and that it plays a role in copper uptake. Taken together with our previous work we hypothesize that Dcytb functions as an intestinal cupric reductase providing Cu+ to Ctr1 for cellular uptake. This work forges a further link between mammalian iron and copper metabolism.

Phillip A. Sharp

Papers

Regulation of Synaptic Structure and Function by FMRP-Associated MicroRNAs miR-125b and miR-132

Duodenal cytochrome b plays a role in copper uptake

Modulation of gene expression by dietary iron

Establishment of a stably transfected cell line to study the function of the ferric reductase Dcytb

A reduction in glucose absorption is observed in the presence of dietary polyphenols using the Caco-2 cell model of human intestinal absorption.