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Expression of the platelet-derived growth factor 2/c- sis gene is highly restricted and controlled at multiple levels. Its
structured mRNA leader, which is unusually long (1022 nucleotides),
serves as a potent translational inhibitor. One of the sites of PDGF2
synthesis is megakaryocytes, implying that PDGF2 translation efficiency
is modulated during megakaryocytic differentiation. To study the role
of the mRNA leader as a translational cis-modulator, the
hybrid T7/vaccinia cytoplasmic expression system was used to disconnect
between determinants controlling transcription, alternative splicing,
and mRNA stability from those controlling translation. Chimeric
transcripts in which the human PDGF2/c- sis mRNA leader
positioned in frame upstream of a reporter gene were used to determine
whether the mRNA leader can confer variable translational efficiencies
during differentiation. It is demonstrated that there is a time window
during megakaryocytic differentiation of K562 cells in which the strong
translational inhibition by PDGF2/c- sis mRNA leader is
relieved. The time course of the translational repression relief is
similar to that of PDGF2/c- sis transcriptional induction
during the differentiation process. A 179-nucleotides CG-rich fragment
immediately upstream of the initiator AUG codon is necessary for
coffering stringent modulation of the translational efficiency. In
NIH3T3 overexpressing translation initiation factor eIF4E, the
inhibitory effect of the mRNA leader of c- sis is not relieved,
suggesting that the changes in the translational machinery during
megakaryocytic differentiation are beyond eIF4E activity. The possible
involvement of a 5`-end-independent translational mechanism is
discussed.
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