The nicotinic acetylcholine receptor subunit Mdα6 from Musca domestica is diversified via post‐transcriptional modification

Abstract

Recent studies showed that deletion of a nicotinic acetylcholine receptor (nAChR) subunit gene, Dalpha6 in Drosophila melanogaster results in a strain that is resistant to spinosad, indicating that Dalpha6 is important for the toxic action of this insecticide. To determine if spinosad resistance in house flies was due to a mutation(s) of Mdalpha6 (the orthologue of Dalpha6 from house flies), cDNAs were cloned and characterized from an insecticide-susceptible and a spinosad-resistant strain of the house fly, Musca domestica. The cDNAs contain a 1470-bp open reading frame encoding 490 amino acid residues, 415-bp 5' untranslated region (UTR) and a polymorphic 3'-UTR of approximately 371 bp. The predicted mature protein possesses 468 amino acid residues, has the typical features of a nAChR alpha subunit and is 97% identical to Dalpha6. Quantitative real-time PCR analysis revealed that Mdalpha6 was expressed in the head and the thorax at 1300- and 26-fold higher levels, respectively, than in the abdomen. There was no difference in the expression level of Mdalpha6 between spinosad-resistant and susceptible strains. Ten isoforms arising from alternative splicing were characterized, with isoform II being most common. A-to-I RNA editing was examined and found at 12 sites: editing at 11 of these sites resulted in an amino acid substitution. Mdalpha6 is linked to autosome 1 (spinosad resistance was previously shown to be linked to autosome 1). Single nucleotide polymorphisms, alternative splicing, mRNA levels and A-to-I RNA editing were compared between head and thorax and between insecticide-susceptible and spinosad-resistant strains. These comparisons indicate that Mdalpha6 is not responsible for spinosad resistance in house flies.

Five nicotinic acetylcholine receptor subunits from the Morotoge shrimp,Pandalopsis japonica: cloning, tissue distribution, and functional expression inXenopusoocytes

“…This may affect the receptor properties as phosphorylation of the TM3-TM4 intracellular loop regulates several aspects of receptor function, including desensitization and aggregation, and may affect the actions of ligands (Borges & Ferns 2001;Hopfield et al 1988;Jones & Sattelle 2010). Different forms generated by alternative splicing have also been observed in the α4 and α6 nAChR subunits (Gao et al 2007;Jones et al 2005;Jones et al 2006;Lansdell & Millar 2000a;Rinkevich & Scott 2009). Some subunits may produce not only isoforms but also truncated transcripts in several insects (Jones et al 2006;Saragoza et al 2003).…”

Unraveling the mystery of spinosad resistance in insects

“…37) In the Caenorhabditis elegans genome, 42 possible nAChR subunits were predicted from which 27 nAChR subunit transcripts have been determined. 36) The relatively small number of nAChR subunits in insects is compensated for by diversification due to alternative exon use and RNA editing 29,[38][39][40] as described below. Alternative splicing plays an important role in expanding protein diversity and is commonly employed to expand diversity of insect nAChRs.…”

“…The biological function of these shortened transcripts is not known, but they have been observed in both D. melanogaster 29) and M. domestica. 38,40) The nAChR with the most alternative transcripts in D. melanogaster is Da6 (two alternative exon 3s and three alternative exon 8s), where five different full length transcripts have been identified. 41) It appears use of alternative transcription may be conserved between species, as the same alternative exon use noted for Da6 was found in M. domestica, 40) and analysis of a6 nAChRs in An.…”

“…38,40) The nAChR with the most alternative transcripts in D. melanogaster is Da6 (two alternative exon 3s and three alternative exon 8s), where five different full length transcripts have been identified. 41) It appears use of alternative transcription may be conserved between species, as the same alternative exon use noted for Da6 was found in M. domestica, 40) and analysis of a6 nAChRs in An. gambiae, Bombyx mori, T. castaneum, Apis mellifera and 12 Drosophila species found all species had two exon 3s (except in A. mellifera) and three exons 8s (except in An.…”

“…We previously identified 12, 13, one and zero A-to-I RNA editing sites in house fly Mda6, Mda5, Mda2, Mdb3, respectively. [38][39][40] The sites of RNA editing for a6 subunit genes are conserved between D. melanogaster, M. domestica and H. virescens. 34,40,41) RNA editing is not randomly distributed across nAChR subunits, nor are the regions of the protein that have resulting amino acid substitutions the same between different subunits.…”