Figure 2.

The 300- and 372nt-long PTCH1b 5'UTRs harbors upstream AUG codons (uAUGs) and open reading frames (uORFs) that impact on associated mRNA translation. (A) Shown is the human sequence of the PTCH1b 5'UTR starting from the 5' portion of the longest reported 372-nucleotide sequence. Three uAUGs (bold and numbered in superscript) are present in the 2 longer sized 5'UTRs. Two of these uAUG would form short uORFs consisting in 40 and 23 codons and sharing the same stop codon (underlined and numbered in superscript) in the 5'UTR. The second uAUG from the 5' end would instead form a longer ORF (89 codons in size), whose stop codon is in the PTCH1b coding sequence and in a different frame (upper case). Starting from 5', arrows mark the beginning of the 372nt, 300nt and 188nt UTR sequence. Hence, the 188nt UTR sequence is included in its entirety in the longer UTRs. (B, C) Introduction of 2 point mutations (c.-248G>T and c.-242A>T) into pGL3-P-type plasmid harboring 300nt long PTCH1b 5'UTR abolishing uAUG¹ and uAUG³ restored luciferase activity to levels comparable to the activity of the plasmid containing the 188nt long 5'UTR (B), with no impact on reporter gene transcription (C). The 372nt 5'UTR behaved similarly to the 300nt one in the reporter and mRNA quantification tests.