Title: An Apparent Pseudo-Exon Acts both as an Alternative Exon That Leads to Nonsense-Mediated Decay and as a Zero-Length Exon
Abstract: AbstractPseudo-exons are intronic sequences that are flanked by apparent consensus splice sites but that are not observed in spliced mRNAs. Pseudo-exons are often difficult to activate by mutation and have typically been viewed as a conceptual challenge to our understanding of how the spliceosome discriminates between authentic and cryptic splice sites. We have analyzed an apparent pseudo-exon located downstream of mutually exclusive exons 2 and 3 of the rat α-tropomyosin (TM) gene. The TM pseudo-exon is conserved among mammals and has a conserved profile of predicted splicing enhancers and silencers that is more typical of a genuine exon than a pseudo-exon. Splicing of the pseudo-exon is fully activated for splicing to exon 3 by a number of simple mutations. Splicing of the pseudo-exon to exon 3 is predicted to lead to nonsense-mediated decay (NMD). In contrast, when "prespliced" to exon 2 it follows a "zero length exon" splicing pathway in which a newly generated 5′ splice site at the junction with exon 2 is spliced to exon 4. We propose that a subset of apparent pseudo-exons, as exemplified here, are actually authentic alternative exons whose inclusion leads to NMD. We thank Larry Chasin and X. H. Zhang for the octamer data set of ESE/ESS values, Clare Gooding for helpful discussions throughout the course of this project and for comments on the manuscript, Andrew Proven and Martin Bootman for the cardiac myocytes, and David Elliott for the kind use of his laboratory to undertake some of the final experiments. We are very grateful to David Grellscheid for invaluable assistance in computational analysis.This study was supported by Wellcome program grant 059879 to C.W.J.S.