THE TRANSLATIONAL REPRESSION MEDIATED BY THE PLATELET-DERIVED GROWTH-FACTOR 2/C-SIS MESSENGER-RNA LEADER IS RELIEVED DURING MEGAKARYOCYTIC DIFFERENTIATION

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.