In vivo RNA–RNA duplexes from human a3 and a5 nicotinic receptor subunit mRNAs Giulia Solda ` a , Silvia Boi a , Stefano Duga a , Diego Fornasari b , Roberta Benfante b , Massimo Malcovati a , Maria Luisa Tenchini a, * a Department of Biology and Genetics for Medical Sciences, University of Milan, via Viotti 3/5, 20133 Milan, Italy b Department of Pharmacology, School of Medicine, University of Milan and CNR Institute of Neuroscience, via Vanvitelli 32, 20129 Milan, Italy Received 12 May 2004; received in revised form 1 November 2004; accepted 6 December 2004 Received by D.L. Court Available online 5 January 2005 Abstract Natural antisense transcripts, because of their potential to form double-stranded RNA (dsRNA) molecules, recently emerged as a mechanism acting on eukaryotic gene regulation at multiple levels. CHRNA3 and CHRNA5, coding for a3 and a5 subunits of the neuronal nicotinic acetylcholine receptor, have been reported to overlap at their 3V ends in human and bovine genomes. In the present paper, four CHRNA3 and three CHRNA5 human transcripts were characterised, leading to the identification of different antisense complementary regions. Since the two genes are coexpressed in some neuronal and non-neuronal tissues, we ventured on the in vivo identification of RNA– RNA duplexes in both humans and cattle. Using an RNase protection-based approach, CHRNA3/CHRNA5 duplexes were detected in human neuroblastoma SY5Y cells, but not in bovine cerebellum. A semi-quantitative analysis of overlapping transcript levels was performed by real-time RT-PCR. Possible consequences of sense-antisense interaction are discussed. D 2004 Elsevier B.V. All rights reserved. Keywords: Natural antisense transcripts (NATs); Neuronal nicotinic acetylcholine receptor (nAChR); Double-stranded RNA; Tail-to-tail overlap; Alternative transcripts 1. Introduction Overlapping genes frequently occur in viral and prokary- otic genomes as well as in mitochondrial DNA, where a dimensional constraint acts on genome size, contributing to the maximisation of the information content of nucleotide sequences (Wagner and Simons, 1994). Gene overlap was therefore considered extremely rare in higher eukaryotes. However, the presence of a surprisingly high number of overlapping genes (more than 8% of all genes) is now emerging in both human and mouse genomes (Yelin et al., 2003; Kiyosawa et al., 2003). The interest on overlapping genes is mainly focused on the gene pairs transcribed from opposite DNA strands, because of their potential to originate natural antisense transcripts (NATs), which can form perfect RNA–RNA duplexes (Kumar and Carmichael, 1998; Van- hee-Brossollet and Vaquero, 1998). Despite the wide range of applications of artificial antisense constructs in basic and clinical research, little is known on the functional role of NATs in regulating eukaryotic gene expression. Moreover, no generalisation concerning the mechanism of action can be drawn based on the few gene pairs that have been experimentally examined. In fact, sense–antisense duplexes 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.12.005 Abbreviations: bp, base pair(s); kb, kilobase(s); CHRNA3, cholinergic receptor nicotinic alpha subunit 3; CHRNA5, cholinergic receptor nicotinic alpha subunit 5; CHRNB4, cholinergic receptor nicotinic beta subunit 4; dsRNA, double-stranded RNA; nAChR, nicotinic acetylcholine receptor; NATs, natural antisense transcripts; UTR, untranslated region; cDNA, DNA, complementary to RNA; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcriptase polymerase chain reaction; RPA, RNase protection assay(s); CNS, central nervous system; EST, expressed sequence tag; ARE, AU-rich element; ADAR, adenosine deaminase that acts on RNA; u, unit(s); dNTP, deoxyribonucleoside triphosphate; DNase, deox- yribonuclease; RNase, ribonuclease; SDS, sodium dodecyl sulfate. * Corresponding author. Tel.: +390250315840; fax: +390250315864. E-mail address: marialuisa.tenchini@unimi.it (M.L. Tenchini). Gene 345 (2005) 155 – 164 www.elsevier.com/locate/gene