The conserved 5'-untranslated leader of Spi-1 (PU.1) mRNA is highly structured and potently inhibits translation in vitro but not in vivo.

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RESUMO

The transcription factor Spi-1 (PU.1) has a central role in regulating myeloid gene expression during hematopoietic development and its overexpression has been implicated in erythroleukemic transformation. Thus regulation of Spi-1 expression has broad significance for hematopoietic development. A comparison of human and murine cDNA sequences demonstrates that the 5'-untranslated region (5'-UTR) of Spi-1 mRNA is as highly conserved as the coding region (87% identical), suggesting that this sequence may be involved in regulating expression of this protein. The experiments presented in this manuscript provide evidence that the 5'-UTR of Spi-1 contains extensive secondary structure, including three stem-loops that precede the AUG codon. Analysis of the in vitro transcribed Spi-1 5'-UTR by partial nuclease digestion sensitivity is consistent with the existence of two of these stem-loops. The 5'-UTR decreased translation of Spi-1 transcripts in reticuloctye lysates 8- to 10-fold. A series of partial deletions of the 5'-UTR identified the sequence corresponding to the stem-loop most proximal to the initiating AUG codon as sufficient for inhibition of translation. However, the effect of the 5'-UTR on translation in vivo was negligible and resulted in only a slight reduction in the number of ribosomes that became associated with the mRNA. Further, this sequence had no affect on expression of luciferase. The disparity between in vivo and in vitro effects, coupled with the observation that endogenous Spi-1 mRNA is wholly associated 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 independent of translation.

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