Analysis of global gene expression: Studies are in progress which have characterized gene expression in the pineal gland. The first stage has involved analysis of the rat pineal gland: The rodent pineal transcriptome was investigated using microarray gene expression. Comparison of midday and midnight expression profiles revealed that a global >2-fold change in the expression of 1000 genes, 2/3 of which increase at night. Among these, 400 increase >4- fold in expression;studies in organ culture reveal that in nearly all cases, the expression of the highly upregulated genes is induced by treatment with NE or cyclic nucleotide analogs. These findings are consistent with the conclusion that NE-cyclic nucleotide signaling is the primary mechanism responsible for the nocturnal increase in gene expression. However, it is also clear that other mechanisms are involved, because a small number of highly rhythmic genes are not induced or are weakly induced by NE treatment. Comparison of the level of gene expression in the pineal gland to the median expression in other tissues indicates that a set of >300 genes are expressed >8- fold higher in the pineal gland. A significant subset of the most highly expressed genes encode proteins involved in melatonin synthesis and the control of this process, including signalling via adrenergic receptors and second messengers including cyclic nucleotides, Ca++ and phospholipids. Clusters of highly expressed genes are associated with the cellular biology of thyroid hormone, retinoid acid, glutamate biology;and, with metal ion homeostasis, membrane trafficking, and the immune response. Other highly and/or rhythmically expressed genes also encode transcription factors, ion channels, transporters, receptors, regulatory molecules and secreted products that have not previously appeared in the pineal literature. Comparison of the pineal gene expression profile to that of several other tissues adds to the evidence that the pineal gland is most similar to the retina by expanding the number of genes that are highly expressed exclusively in these two tissues. This study indicates that control of pineal biology is significantly more complex than previously thought, that the number of highly expressed genes in the pineal gland and retina is higher than previously thought, and also provides molecular evidence to suspect that the gland might function outside of the highly conserved role it plays in melatonin production. The work on the rodent pineal gland is being followed up with similar work on the pineal gland of the monkey and human, so as to determine the similarity of the patterns of gene expression in these three tissues. This work is being extended using RNA Seq technology, with focus on miRNA and long noncoding RNAs in addition to annotated genes. Long noncoding RNAs (From Coon et al, PNAS, 2012): Long noncoding RNAs (lncRNAs) play a broad range of biological roles, including regulation of expression of genes and chromosomes. Here, we present evidence that lncRNAs are involved in vertebrate circadian biology. Differential night/day expression of 112 lncRNAs (0.3 to >50 kb) occurs in the rat pineal gland, which is the source of melatonin, the hormone of the night. Approximately one-half of these changes reflect nocturnal increases. Studies of eight lncRNAs with 2- to >100-fold daily rhythms indicate that, in most cases, the change results from neural stimulation from the central circadian oscillator in the suprachiasmatic nucleus (doubling time = 0.5-1.3 h). Light exposure at night rapidly reverses (halving time = 9-32 min) levels of some of these lncRNAs. Organ culture studies indicate that expression of these lncRNAs is regulated by norepinephrine acting through cAMP. These findings point to a dynamic role of lncRNAs in the circadian system. MicroRNAs: MicroRNAs (miRNAs) play a broad range of roles in biological regulation. In this study rat pineal miRNAs were profiled for the first time and their importance evaluated by focusing on the main function of the pineal gland, melatonin synthesis. Next-generation sequencing and related methods revealed the miRNA population is dominated by a small group of miRNAs: 75% is accounted for by 10 miRNAs;miR-182 represents 28%. In addition to miR-182, miR-183 and miR-96 are also highly enriched in the pineal gland, a distinctive pattern also found in the retina. This effort also identified previously unrecognized miRNAs and other small non-coding RNAs. Pineal miRNAs do not exhibit a marked night/day difference in abundance with few exceptions (eg. 2-fold night/day differences in the abundance of miR-96 and miR-182);this contrasts sharply with the dynamic 24-hour pattern that characterizes the pineal transcriptome. During development, the abundance of most pineal-enriched miRNAs increases;however, there is a marked decrease in at least one, miR-483. miR-483 is a likely regulator of melatonin synthesis, based on the following: it inhibits melatonin synthesis by pinealocytes in culture;it acts via predicted binding sites in the 3-prime UTR of arylalkylamine N-acetyltransferase (Aanat), the penultimate enzyme in melatonin synthesis;and, it exhibits a developmental profile opposite to that of Aanat transcripts. These observations support the hypothesis that miR-483 suppresses Aanat mRNA levels during development and that the developmental decrease in miR-483 abundance promotes melatonin synthesis.

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6
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2012
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$470,934
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