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Prader-Willi syndrome (PWS) is caused by a loss of paternally expressed genes in an imprinted region of chromosome 15q. Among the canonical PWS phenotypes are hyperphagic obesity, central hypogonadism, and low growth hormone (GH). Rare microdeletions in PWS patients define a 91-kb minimum critical deletion region encompassing 3 genes, including the noncoding RNA gene SNORD116. Here, we found that protein and transcript levels of nescient helix loop helix 2 (NHLH2) and the prohormone convertase PC1 (encoded by PCSK1) were reduced in PWS patient induced pluripotent stem cell-derived (iPSC-derived) neurons. Moreover, Nhlh2 and Pcskl expression were reduced in hypothalami of fasted Snord116 paternal knockout ([Snord116.sup.p/m+]) mice. Hypothalamic Agrp and Npy remained elevated following refeeding in association with relative hyperphagia in Snord116p/m+ mice. Nhlh2-deficient mice display growth deficiencies as adolescents and hypogonadism, hyperphagia, and obesity as adults. Nhlh2 has also been shown to promote Pcsk1 expression. Humans and mice deficient in PC1 display hyperphagic obesity, hypogonadism, decreased GH, and hypoinsulinemic diabetes due to impaired prohormone processing. Here, we found that [Snord116.sup.p/m+] mice displayed in vivo functional defects in prohormone processing of proinsulin, pro-GH-releasing hormone, and proghrelin in association with reductions in islet, hypothalamic, and stomach PC1 content. Our findings suggest that the major neuroendocrine features of PWS are due to PC1 deficiency., Introduction Prader-Willi syndrome (PWS) is the most common syndromic obesity, affecting 1 in 25,000 live births (1, 2). PWS results from a loss of paternally expressed genes at 15q11.2-q13 (Figure [...]

A-to-I RNA editing is an important process for generating molecular diversity in the brain through modification of transcripts encoding several proteins important for neuronal signaling. We investigated the relationships between the extent of editing at multiple substrate transcripts (5HT2C, MGLUR4, CADPS, GLUR2, GLUR4, and GABRA3) in brain tissue obtained from adult humans and rhesus macaques. Several patterns emerged from these studies revealing conservation of editing across primate species. Additionally, variability in the human population allows us to make novel inferences about the co-regulation of editing at different editing sites and even across different brain regions. [ABSTRACT FROM AUTHOR]

The article reports on the RNA editing as a tool in therapeutic target for central nervous systems (CNS) disorders. This study looked into the conversion of adenosine to inosine by RNA editing which genomically encodes sequences. Changes in RNA editing may be the cause in human brain disorders. The role of adenosine deaminases acting on RNA is discussed further.

Abstract: The serotonin 2C receptor (5-HT2CR) plays a significant role in psychiatric disorders (e.g., depression) and is a target for pharmacotherapy. The 5-HT2CR is widely expressed in brain and spinal cord and is the only G-protein coupled receptor currently known to undergo mRNA editing, a post-transcriptional modification that results in translation of distinct, though closely related, protein isoforms. The 5-HT2CR RNA can be edited at five sites to alter up to three amino acids resulting in modulation of receptor:G-protein coupling and constitutive activity. To rapidly quantify changes ex vivo in individual 5-HT2CR isoform levels in response to treatment, we adapted quantitative (real-time) reverse transcription polymerase chain reaction (qRT-PCR) utilizing TaqMan® probes modified with a minor groove binder (MGB). Probes were developed for four 5-HT2CR RNA isoforms and their sensitivity and specificity were validated systematically using standard templates. Relative expression of the four isoforms was measured in cDNAs from whole brain extracted from 129S6 and C57BL/6J mice. Rank order derived from this qRT-PCR analysis matched that derived from DNA sequencing. In mutant mice solely expressing either non-edited or fully edited 5-HT2CR transcripts, only expected transcripts were detected. These data suggest this qRT-PCR method is a precise and rapid means to detect closely related mRNA sequences ex vivo without the necessity of characterizing the entire 5-HT2CR profile. Implementation of this technique will expand and expedite studies of specific brain 5-HT2CR mRNA isoforms in response to pharmacological, behavioral and genetic manipulation, particularly in ex vivo studies which require rapid collection of data on large numbers of samples. [Copyright &y& Elsevier]

RNA editing is a post-transcriptional modification in which adenosine residues are converted to inosine (adenosine-to-inosine editing). Commonly used methodologies to quantify RNA editing levels involve either direct sequencing or pyrosequencing of individual cDNA clones. The limitations of these methods lead to a small number of clones characterized in comparison to the number of mRNA molecules in the original sample, thereby producing significant sampling errors and potentially erroneous conclusions. We have developed an improved method for quantifying RNA editing patterns that increases sequence analysis to an average of more than 800,000 individual cDNAs per sample, substantially increasing accuracy and sensitivity. Our method is based on the serotonin 2C receptor (5-hydroxytryptamine(2C); 5HT(2C)) transcript, an RNA editing substrate in which up to five adenosines are modified. Using a high-throughput multiplexed transcript analysis, we were able to quantify accurately the expression of twenty 5HT(2C) isoforms, each representing at least 0.25% of the total 5HT(2C) transcripts. Furthermore, this approach allowed the detection of previously unobserved changes in 5HT(2C) editing in RNA samples isolated from different inbred mouse strains and dissected brain regions, as well as editing differences in alternatively spliced 5HT(2C) variants. This approach provides a novel and efficient strategy for large-scale analyses of RNA editing and may prove to be a valuable tool for uncovering new information regarding editing patterns in specific disease states and in response to pharmacological and physiological perturbation, further elucidating the impact of 5HT(2C) RNA editing on central nervous system function.

Transcripts encoding 5-HT(2C) receptors are modified posttranscriptionally by RNA editing, generating up to 24 protein isoforms. In recombinant cells, the fully edited isoform, 5-HT(2C-VGV), exhibits blunted G-protein coupling and reduced constitutive activity. The present studies examine the signal transduction properties of 5-HT(2C-VGV) receptors in brain to determine the in vivo consequences of altered editing. Using mice solely expressing the 5-HT(2C-VGV) receptor (VGV/Y), we demonstrate reduced G-protein coupling efficiency and high-affinity agonist binding of brain 5-HT(2C-VGV) receptors. However, enhanced behavioral sensitivity to a 5-HT(2C) receptor agonist was also seen in mice expressing 5-HT(2C-VGV) receptors, an unexpected finding given the blunted G-protein coupling. In addition, mice expressing 5-HT(2C-VGV) receptors had greater sensitivity to a 5-HT(2C) inverse agonist/antagonist enhancement of dopamine turnover relative to wild-type mice. These behavioral and biochemical results are most likely explained by increases in 5-HT(2C) receptor binding sites in the brains of mice solely expressing 5-HT(2C-VGV) receptors. We conclude that 5-HT(2C-VGV) receptor signaling in brain is blunted, but this deficiency is masked by a marked increase in 5-HT(2C) receptor binding site density in mice solely expressing the VGV isoform. These findings suggest that RNA editing may regulate the density of 5-HT(2C) receptor binding sites in brain. We further caution that the pattern of 5-HT(2C) receptor RNA isoforms may not reflect the pattern of protein isoforms, and hence the inferred overall function of the receptor.