Mice with altered serotonin 2C receptor RNA editing display characteristics of Prader–Willi syndrome
Introduction
The conversion of adenosine to inosine (A-to-I) by RNA editing is a widespread RNA processing event by which genomically encoded sequences are altered through the site-specific deamination of adenosine residue(s) in precursor and mature mRNA transcripts (Gott and Emeson, 2000). The majority of well-characterized A-to-I editing events involve non-synonymous codon changes in RNAs encoding proteins involved in nervous system function including ligand- and voltage-gated ion channels, a G-protein coupled receptor and components of the synaptic release machinery (Burns et al., 1997, Gott and Emeson, 2000, Hoopengardner et al., 2003). Transcripts encoding the 5HT2C receptor can be modified by five A-to-I editing events (sites A–E) to generate as many as 24 protein isoforms that differ by up to three amino acids within the predicted second intracellular loop of the receptor, a region involved in receptor:G-protein coupling (Burns et al., 1997, Pin et al., 1994). Though initial sequence analyses of cDNAs isolated from dissected rat and human brains predicted the region-specific expression of as many as 12 major 5HT2C receptor isoforms encoded by eighteen distinct RNA species (Burns et al., 1997, Niswender et al., 1999), more recent studies identified the expression of 26 of the 32 possible mRNA isoforms and determined that only 4–6 of these mRNAs represent more than 5% of total 5HT2C transcripts in rats and humans, respectively (Dracheva et al., 2009). Alterations in 5HT2C receptor editing have been observed in suicide victims with a history of major depression, schizophrenia, or bipolar disorder (Dracheva et al., 2008, Gurevich et al., 2002, Iwamoto and Kato, 2003, Niswender et al., 2001) and in response to antidepressant and antipsychotic treatment (Englander et al., 2005, Gurevich et al., 2002, Sodhi et al., 2005). The fully-edited (VGV) isoform of the human 5HT2C receptor, encoding valine, glycine and valine at amino acid positions 156, 158 and 160, respectively, exhibits reduced constitutive activity and decreased G-protein coupling efficacy when compared to the genomically encoded (INI) isoform in heterologous expression systems (Berg et al., 2001, Burns et al., 1997, Fitzgerald et al., 1999, Niswender et al., 1999, Wang et al., 2000), yet the physiologic relevance of 5HT2C RNA editing in nervous system function remains unclear.
Prader–Willi syndrome is a maternally imprinted human disorder resulting from a loss of paternal gene expression on chromosome 15q11–13 that is characterized by a complex phenotype including cognitive deficits, infantile hypotonia and failure to thrive, short stature, hypogonadism and hyperphagia which can lead to morbid obesity (Goldstone, 2004, Nicholls and Knepper, 2001). Multiple mouse models with deficiencies of one or more PWS candidate genes have partially correlated individual genes with aspects of the PWS phenotype (Bischof et al., 2007, Cattanach et al., 1992, Ding et al., 2008, Gabriel et al., 1999, Gerard et al., 1999, Muscatelli et al., 2000, Skryabin et al., 2007, Tsai et al., 1999b, Yang et al., 1998). Among these imprinted candidate genes are the brain-specific small nucleolar RNAs (snoRNAs), HBII-13, HBII-85 and HBII-52 (Cavaille et al., 2000). HBII-52 (SNORD115), and its mouse orthologue (MBII-52; Snord115), are members of the box C/D family of snoRNAs that are responsible for directing the 2′-O-methylation of specific ribose moieties in pre-ribosomal RNA transcripts and U small nuclear RNAs (Kiss, 2002). HBII-52/MBII-52 is complementary to an 18 nucleotide segment of 5HT2C mRNA containing three of five editing sites (E, C and D), predicting 2′-O-methylation of the ribose for the adenosine nucleoside at the C-site (Cavaille et al., 2000), an editing position that can significantly affect the function of encoded 5HT2C protein isoforms (Burns et al., 1997, Niswender et al., 1999). HBII-52/MBII-52 also has been observed to affect 5HT2C RNA editing/splicing patterns using tissue culture model systems (Kishore and Stamm, 2006, Vitali et al., 2005). Analyses of 5HT2C transcripts have indicated that site-specific editing is increased in brain samples from both PWS patients (Kishore and Stamm, 2006) and a mouse model of PWS (PWS-ICdel) that further exhibits deficits in specific 5HT2C-mediated behaviors (Doe et al., 2009). Though these studies have correlated changes in both 5HT2C receptor expression and function in the absence of 15q11–13 gene expression, here we show that increased 5HT2C RNA editing in mutant mice recapitulates many aspects of this disorder, suggesting an important role for altered 5HT2C function in the etiology of Prader–Willi syndrome.
Section snippets
Generation of 5HT2C-VGV mice
A genomic fragment containing a portion of the mouse 5HT2C gene was isolated from a 129S6 BAC library (Genome Systems, St. Louis, MO) and a 7.4-kb AvrII restriction fragment containing exon 5 and a portion of the flanking introns was subcloned into a modified pBKSII- vector (Stratagene, La Jolla, CA). The five edited adenosine residues within exon 5 were mutated to guanosine moieties using overlap-extension PCR (Ho et al., 1989). A DNA fragment encoding loxP-flanked neomycin phosphotransferase,
Generation of mutant mice solely expressing the fully-edited (VGV) isoform of the 5HT2C receptor
To investigate the importance of normal 5HT2C RNA editing patterns, we generated mutant mice solely expressing the fully edited isoform of the receptor (5HT2C-VGV). We chose this receptor isoform based upon its decreased G-protein coupling efficacy and lack of constitutive activity compared to other 5HT2C isoforms (Berg et al., 2001, Burns et al., 1997, Fitzgerald et al., 1999, Niswender et al., 1999, Wang et al., 2000). Mice solely expressing 5HT2C-VGV receptors were generated by homologous
Discussion
The editing of 5HT2C transcripts modulates multiple aspects of 5HT2C receptor signaling and expression in heterologous systems (Berg et al., 2001, Burns et al., 1997, Fitzgerald et al., 1999, Flomen et al., 2004, Marion et al., 2004, Niswender et al., 1999, Price et al., 2001, Wang et al., 2000), yet the physiologic importance for the existence of multiple 5HT2C isoforms has not been fully explored. Recent studies by Kawahara et al. (2008) have characterized mutant mice comparable to those
Acknowledgments
We thank Dr. Douglas McMahon and Christopher Ciarleglio for assistance with locomotor activity, Ryan Strachan, Michael Hughes, James Gilbert, Li Peng and Apoorwa Thati for technical assistance, and Drs. Tim Nagy and Maria Johnson of the UAB Small Animal Phenotyping Core for metabolic measurements. We also thank Drs. Elisabeth Dykens, Pat Levitt, and Larry Zwiebel for critical reading of the manuscript. This work was supported by grants from the National Institutes of Health (NS35891 to R.B.E.;
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Present address: Department of Life Sciences, Winston-Salem State University, Winston-Salem, NC 27110, USA.