Effect of TPO gene mutations on the composition of uORFs in TPO mRNA.

TPO transcripts starting from the main promoter in exon 2 are shown. Boxes numbered in italics represent exons. The uORFs are drawn as thick red lines and are placed into one of the three reading frames (+1, 0, and −1). The TPO coding region is shown as a thick blue arrow. Numbers indicate the order in which the uAUGs appear in the full-length TPO mRNA (therefore, uAUGs 1 through 4 are not shown). The eighth AUG is the physiological initiation codon. (A) Translation of normal TPO mRNA is physiologically almost completely inhibited by the presence of uORFs in the 5′-UTR. In particular, the uORF 7 is a potent inhibitor of translation, most likely because of its extension beyond the physiological start site. (B) A splice donor mutation in the Dutch HT family causes exon 3 skipping (ΔE3) that deletes uORF7 and shifts the TPO coding sequence into reading frame +1. TPO translation now initiates from the fifth and sixth AUGs. (C) The Japanese mutation I consists of a single G nucleotide deletion (ΔG) that shifts the TPO coding sequence into reading frame −1. TPO translation now initiates from the seventh AUG. Note that both the Dutch and the Japanese mutation I create altered TPO signal peptides, but do not alter the sequence of the mature TPO protein. Both signal peptides remain functionally active and promote secretion of a biologically active TPO protein. (D) The Japanese mutation II creates a premature stop codon in uORF7. This allows reinitiation of translation at the physiological start site (the eighth AUG).