Nucleic Acids Research, Vol 25, Issue 14 2869-2876, Copyright © 1997 by Oxford University Press
JO Hensold, CA Stratton and D Barth
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.
ARTICLES
The conserved 5'-untranslated leader of Spi-1 (PU.1) mRNA is highly structured and potently inhibits translation in vitro but not in vivo
The University/Ireland Cancer Center, Department of Medicine and Case Western Reserve University, Cleveland, OH 44106, USA. joh2@po.cwru.edu
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