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

Abstract

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 out in a fraction of its mRNA. To characterize the molecular mechanism underlying this alternative splicing, minigenes of TPO genomic constructs with variable exon-intron configurations or carrying exclusively the TPO cDNA were generated and transiently transfected in the Hep3B cell line. We have found that the final rate of the alternative intron splicing is determined by three elements: the presence of upstream constitutive introns, the suboptimal splice sites of the alternative intron and the length of the alternative intron itself. Our results indicate that the recognition of suboptimal intra-exonic splice junctions in the TPO gene is influenced by the assembly of the spliceosome complex on constitutive introns and by a qualitative scanning of the sequence by the transcriptional/splicing machinery complex primed by upstream splicing signals.

Complex splicing pattern generates great diversity in human NF1 transcripts

“…This finding underlines the importance of the acceptor site in splice site selection. This is in agreement with a recent study [22] in which it was shown that mutating the acceptor site to a stronger site was more effective for full intron splicing of an alternative intron, than strengthening the donor site. Likewise, there was an inverse relationship found between the length of the polythimidine tract, which influences the acceptor strength, at the exon 9 acceptor site and the proportion of exon 9 deleted CFTR mRNA transcripts [23].…”

Regulation of Alternative Splicing Through Coupling with Transcription and Chromatin Structure

“…In genes in which multiple alternative exons exist, coordination of splicing events favors some specific combinations over others. Although in many cases alternative splicing events are supposed to occur independently, some specific cases of coordination have been reported www.annualreviews.org • Regulation of Alternative Splicing (62)(63)(64), and a statistical analysis of ESTs (expressed sequence tags) suggests that it might be more common than generally thought (63). Coordination could also arise in posttranscriptional splicing if, for example, different events are regulated by a shared set of splicing factors.…”

The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript

“…In vivo cooperation between introns for their processing has been previously observed, but the mechanism has remained unclear (Neel et al 1993;Nesic and Maquat 1994;Romano et al 2001). For instance, introns of the tumor necrosis factor b (TNFb) transcript are spliced more efficiently in HeLa cells when upstream introns are still present (Neel et al 1993).…”