The α human folate receptor (αhFR), or KB cell folate receptor, gene contains two major promoters that produce transcripts, KB1 and KB4, varying only in the length and sequence of their 5′ untranslated regions (UTRs). Using RNase protection assays specific for each isoform, we show that the level of expression of these two transcripts is tissue-specific, indicating that promoter usage is regulated, not constitutive. RNA stabilities and translational efficiencies of the KB1 and KB4 transcripts were compared to determine the functional significance of the different 5′ UTRs. Analyses of RNA turnover in vivo with actinomycin D to block new transcription and in vitro with a cytoplasmic extract indicate no discernible differences in the stabilities of the two transcripts. However, the KB4 transcript is 2–3-fold more efficiently translated in wheat germ extracts in vitro and transfected CHO cells in vivo. Also, high ionic strength, which favours the formation of RNA secondary structure, differentially affects the translational efficiencies of the two transcripts. Translation of the longer KB1 mRNA is 2–5-fold more inhibited by hypertonic conditions than translation of the KB4 mRNA. Because the 5′ UTR of KB1 is approximately four times longer than the 5′ UTR of KB4, 149 bp (75%) of the KB1 5′ UTR were deleted to determine whether the long leader sequence inhibited translation. The resulting derivative, dKB1, has a 5′ UTR similar in length, but not sequence, to the 5′ UTR of KB4. dKB1 is translated at a level approaching that of KB4 in wheat germ extracts, indicating that the upstream portion of the 5′ leader sequence contributes to the relative translational inefficiency of KB1. Hence, one consequence of tissue-specific promoter usage is the production of αhFR transcripts with different 5′ non-coding regions that affect translational efficiency.
- The Biochemical Society, London © 1997