Splicing of constitutive upstream introns is essential for the recognitionof intra-exonic suboptimal splice sites in the thrombopoietin gene

The human thrombopoietin (TPO) gene, which codes for the principal cytokine involved in platelet maturation, shows a peculiar alternative splicing of its last exon, where an intra-exonic 116 nt alternative intron is spliced o ut in: a fraction of its mRNA, To characterize the molecular mechanism unde rlying this alternative splicing, minigenes of TPO genomic constructs with variable exon-intron configurations or carrying exclusively the TPO cDNA we re generated and transiently transfected in the Hep3B cell line. We have fo und that the final rate of the alternative intron splicing is determined by three elements: the presence of upstream constitutive introns, the subopti mal splice sites of the alternative intron and the length of the alternativ e intron itself, Our results indicate that the-recognition of suboptimal in tra-exonic splice junctions in the TPO gene is influenced by the assembly o f the spliceosome complex on constitutive introns and by a qualitative scan ning of the sequence by-the transcriptional/splicing machinery complex prim ed by upstream splicing signals.