Analysis of the translational efficiency of TPO P2-wt transcripts in vitro and in transfected COS cells. (A) Exon composition of the TPO mRNA. The TPO protein coding region is shown in gray. The uAUG triplets (filled circles) are numbered in the order they appear in the P1-wt transcript and the resulting ORFs (horizontal solid lines) are placed in the three possible reading frames (Roman numerals). The thick solid line with arrowhead represents the ORF encoding TPO protein. (B) In vitro transcription translation analysis. Equal amounts of in vitro transcribed TPO mRNA variants (top) were translated in vitro in reticulocyte lysate in the presence of35S-methionine (bottom). ▵UTR, mRNA with deletion of the entire 5′-UTR; nm, mRNA with no mutations in the 5′-UTR; numbers above individual lanes indicate the position of mutated uAUGs. The protein bands in the bottom panel were identified as: wt TPO, initiation at the physiological start site (AUG 8); asterisk, cryptic non-AUG initiation within exon 3. (C) TPO production and secretion by transfected COS cells. Presence of TPO in COS cell supernatants was determined by bioassay. Mock transfected COS cells were set as background and cells transfected with a ▵UTR expression construct as 100%. Bars indicate the median ± SEM of triplicates.
