Nucleic Acids Research Advance Access originally published online on March 4, 2007
Nucleic Acids Research 2007 35(6):1947-1957; doi:10.1093/nar/gkm062
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Nucleic Acids Research, 2007, Vol. 35, No. 6 1947-1957
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Genomics |
Beyond the 3' end: experimental validation of extended transcript isoforms
INSERM ERM206, Université de la Méditerranée, Luminy case 928, 13288 Marseille cedex 09, France
*To whom correspondence should be addressed. Tel: 33 (0)1 69 15 46 32; Fax: 33 (0)1 69 15 46 29; Email: gautheret{at}tagc.univ-mrs.fr
Received November 22, 2006. Accepted January 19, 2007.
High throughput EST and full-length cDNA sequencing have revealed extensive variations at the 3' ends of mammalian transcripts. Whether all of these changes are biologically meaningful has been the subject of controversy, as such, results may reflect in part transcription or polyadenylation leakage. We selected here a set of tandem poly(A) sites predicted from EST/cDNA sequence analysis that (i) are conserved between human and mouse, (ii) produce alternative 3' isoforms with unusual size features and (iii) are not documented in current genome databases, and we submitted these sites to experimental validation in mouse tissues. Out of 86 tested poly(A) sites from 44 genes, 84 were individually confirmed using a specially devised RT-PCR strategy. We then focused on validating the exon structure between distant tandem poly(A) sites separated by over 3 kb, and between stop codons and alternative poly(A) sites located at 4.5 kb or more, using a long-distance RT-PCR strategy. In most cases, long transcripts spanning the whole poly(A)–poly(A) or stop-poly(A) distance were detected, confirming that tandem sites were part of the same transcription unit. Given the apparent conservation of these long alternative 3' ends, different regulatory functions can be foreseen, depending on the location where transcription starts.
Present address: Takeshi Ara, Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818 – Japan Daniel Gautheret, Univ. Paris Sud, CNRS IGM, Bat 400, 91400 Orsay, France
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