CONSERVED 5'-UNTRANSLATED LEADER OF SPI-1 (PU.1) MESSENGER-RNA IS HIGHLY STRUCTURED AND POTENTLY INHIBITS TRANSLATION IN-VITRO BUT NOT IN-VIVO

The transcription factor Spi-1 (PU.1) has a central role in regulating myeloid gene expression during hematopoietic development and its over expression has been implicated in erythroleukemic transformation, Thus regulation of Spi-1 expression has broad significance for hematopoiet ic development, A comparison of human and murine cDNA sequences demons trates that the 5'-untranslated region (5'-UTR) of Spi-1 mRNA is as hi ghly conserved as the coding region (87% identical), suggesting that t his sequence may be involved in regulating expression of this protein, The experiments presented in this manuscript provide evidence that th e 5'-UTR of Spi-1 contains extensive secondary structure, including th ree stem-loops that precede the AUG codon, Analysis of the in vitro tr anscribed Spi-1 5'-UTR by partial nuclease digestion sensitivity is co nsistent with the existence of two of these stem-loops, The 5'-UTR dec reased translation of Spi-1 transcripts in reticulocyte lysates 8- to 10-fold, A series of partial deletions of the 5'-UTR identified the se quence corresponding to the stem-loop most proximal to the initiating AUG codon as sufficient for inhibition of translation, However, the ef fect of the 5'-UTR on translation in vivo was negligible and resulted in only a slight reduction in the number of ribosomes that became asso ciated with the mRNA, Further, this sequence had no affect on expressi on of luciferase. The disparity between in vivo and in vitro effects, coupled with the observation that endogenous Spi-1 mRNA is wholly asso ciated with polysomes in MEL cells, suggests that additional cellular mechanisms contribute to regulation of Spi-1 expression in these cells or that conservation of these sequences serves a function that is ind ependent of translation.