Genome-scale analysis of human mRNA 5′ coding sequences based on expressed sequence tag (EST) database

The “5′ end mRNA artifact” issue refers to the incorrect assignment of the first AUG codon in an mRNA, due to the incomplete determination of its 5′ end sequence. We performed a systematic identification of coding regions at the 5′ end of all human known mRNAs, using an automated expressed sequence tag (EST)-based approach. Following parsing of more than 7million BLAT alignments, we found 477 human loci, out of 18,665 analyzed, in which an extension of the mRNA 5′ coding region was identified. Proof-of-concept confirmation was obtained by in vitro cloning and sequencing for GNB2L1, QARS and TDP2 cDNAs, and the consequences for the functional studies of these loci are discussed. We also generated a list of 20,775 human mRNAs where the presence of an in-frame stop codon upstream of the known start codon indicates completeness of the coding sequence at 5′ in the current form. ► Start codon may be incorrectly assigned if mRNA sequence is not complete at 5′ end. ► An EST-based computational approach identifies extended 5′ coding regions. ► The mRNA 5′ coding region is extended in 477 human loci out of 18,665 analyzed. ► Experimental confirmation has been obtained for GNB2L1, QARS and TDP2. ► 20,775 human mRNAs have a coding region not further extendable by EST sequences.