Sex-specific effect of P2Y2 purinergic receptor on glucose metabolism during acute inflammation.

The sex of an animal impacts glucose sensitivity, but little information is available regarding the mechanisms causing that difference, especially during acute inflammation. We examined sex-specific differences in the role of the P2Y2 receptor (P2Y2R) in glucose flux with and without LPS challenge. Male and female wild-type and P2Y2R knockout mice (P2Y2R-/-) were injected with LPS or saline and glucose tolerance tests (GTT) were performed. P2Y2R, insulin receptor, and GLUT4 transporter gene expression was also evaluated. Female mice had reduced fasting plasma glucose and females had reduced glucose excursion times compared to male mice during GTT. P2Y2R-/- males had significantly decreased glucose flux throughout the GTT as compared to all female mice. Acute inflammation reduced fasting plasma glucose and the GTT area under the curve in both sexes. While both wild-type and P2Y2R-/- male animals displayed reduced fasting glucose in LPS treatment, female mice did not have significant difference in glucose tolerance, suggesting that the effects of P2Y2R are specific to male mice, even under inflammatory conditions. Overall, we conclude that the role for the purinergic receptor, P2Y2R, in regulating glucose metabolism is minimal in females but plays a large role in male mice, particularly in the acute inflammatory state.

Using mouse models to unlock the secrets of non-synonymous RNA editing.

The deamination of adenosine to inosine by RNA editing is a widespread post-transcriptional process that expands genetic diversity. Selective substitution of inosine for adenosine in pre-mRNA transcripts can alter splicing, mRNA stability, and the amino acid sequence of the encoded protein. The functional consequences of RNA editing-dependent amino acid substitution are known for only a handful of RNA editing substrates. Many of these studies began in heterologous mammalian expression systems; however, the gold-standard for determining the functional significance of transcript-specific re-coding A-to-I editing events is the generation of a mouse model that expresses only one RNA editing-dependent isoform. The frequency of site-specific RNA editing varies spatially, temporally, and in some diseases, therefore, determining the profile of RNA editing frequency is also an important element of research. Here we review the strengths and weaknesses of existing mouse models for the study of RNA editing, as well as methods for quantifying RNA editing frequencies in vivo. Importantly, we highlight opportunities for future RNA editing studies in mice, projecting that improvements in genome editing and high-throughput sequencing technologies will allow the field to excel in coming years.

One hundred million adenosine-to-inosine RNA editing sites: hearing through the noise.

The most recent work toward compiling a comprehensive database of adenosine-to-inosine RNA editing events suggests that the potential for RNA editing is much more pervasive than previously thought; indeed, it is manifest in more than 100 million potential editing events located primarily within Alu repeat elements of the human transcriptome. Pairs of inverted Alu repeats are found in a substantial number of human genes, and when transcribed, they form long double-stranded RNA structures that serve as optimal substrates for RNA editing enzymes. A small subset of edited Alu elements has been shown to exhibit diverse functional roles in the regulation of alternative splicing, miRNA repression, and cis-regulation of distant RNA editing sites. The low level of editing for the remaining majority may be non-functional, yet their persistence in the primate genome provides enhanced genomic flexibility that may be required for adaptive evolution.

High-throughput multiplexed transcript analysis yields enhanced resolution of 5-hydroxytryptamine 2C receptor mRNA editing profiles.

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.

Puf1p acts in combination with other yeast Puf proteins to control mRNA stability.

The eukaryotic Puf proteins bind 3' untranslated region (UTR) sequence elements to regulate the stability and translation of their target transcripts, and such regulatory events are critical for cell growth and development. Several global genome analyses have identified hundreds of potential mRNA targets of the Saccharomyces cerevisiae Puf proteins; however, only three mRNA targets for these proteins have been characterized thus far. After direct testing of nearly 40 candidate mRNAs, we established two of these as true mRNA targets of Puf-mediated decay in yeast, HXK1 and TIF1. In a novel finding, multiple Puf proteins, including Puf1p, regulate both of these mRNAs in combination. TIF1 mRNA decay can be stimulated individually by Puf1p and Puf5p, but the combination of both proteins is required for full regulation. This Puf-mediated decay requires the presence of two UGUA binding sites within the TIF1 3' UTR, with one site regulated by Puf5p and the other by both Puf1p and Puf5p. Alteration of the UGUA site in the tif1 3' UTR to more closely resemble the Puf3p binding site broadens the specificity to include regulation by Puf3p. The stability of the endogenously transcribed HXK1 mRNA, cellular levels of Hxk1 protein activity, and HXK1 3' UTR-directed decay are affected by Puf1p and Puf5p as well as Puf4p. Together these results identify the first mRNA targets of Puf1p-mediated decay, describe similar yet distinct combinatorial control of two new target mRNAs by the yeast Puf proteins, and suggest the importance of direct testing to evaluate RNA-regulatory mechanisms.