Cryptic splicing sites are differentially utilized in vivo.

It has long been considered that cryptic splice sites are ignored by the splicing machinery in the context of intact genuine splice sites. In the present study, it is shown that cryptic splice sites are utilized in all circumstances, when the authentic site is intact, partially functional or completely abolished. Their use would therefore contribute to a background lack of fidelity in the context of the wild-type sequence. We also found that a mutation at the 5' splice site of beta-globin intron 1 accommodates multiple cryptic splicing pathways, including three previously reported pathways. Focusing on the two major cryptic 5' splice sites within beta-globin exon 1, we show that cryptic splice site selection ex vivo varies depending upon: (a) the cell stage of development during terminal erythroid differentiation; (b) the nature of the mutation at the authentic 5' splice site; and (c) the nature of the promoter. Finally, we found that the two major cryptic 5' splice sites are utilized with differential efficiencies in two siblings sharing the same beta-globin chromosome haplotype in the homozygous state. Collectively, these data suggest that intrinsic, sequence specific factors and cell genetic background factors both contribute to promote a subtle differential use of cryptic splice sites in vivo.

Spi-1/PU.1 but not Fli-1 inhibits erythroid-specific alternative splicing of 4.1R pre-mRNA in murine erythroleukemia cells.

The inclusion of exon 16 in mature protein 4.1R mRNA arises from a stage-specific splicing event that occurs during late erythroid development. We have shown that mouse erythroleukemia (MEL) cells reproduce this erythroid-specific splicing event upon induction of differentiation. We here found that this splicing event is regulated specifically in erythroleukemic cells that have the potential to differentiate and produce hemoglobin, regardless of the nature of the differentiation inducer. Knowing that dysregulated expression of spi-1/pu.1 and fli-1 oncogenes is involved in MEL cell differentiation arrest, we looked at their effect on exon 16 erythroid splicing. We found that exon 16 inclusion requires Spi-1/PU.1 shutdown in MEL cells, and that enforced expression of Spi-1/PU.1 inhibits exon selection, regardless of the presence or absence of a chemical inducer. By contrast, endogenous overexpression or enforced expression of Fli-1 has no effect on exon selection. We further showed that Spi-1/PU.1 acts similarly on the endogenous and on a transfected exon 16, suggesting a promoter-independent effect of Spi-1/PU.1 on splicing regulation. This study provides the first evidence that Spi-1/PU.1 displays the unique property, not shared with Fli-1, to inhibit erythroid-specific pre-mRNA splicing in erythroleukemia cell context.

HnRNP A1 tethers KSRP to an exon splicing silencer that inhibits an erythroid-specific splicing event in PU.1-induced erythroleukemia.

Exon 16 inclusion is a critical splicing event that triggers the production of a functional protein 4.1R in mature normal erythroblasts, and is obviated in PU.1-induced erythroleukemia cells. Exon 16 contains an exonic splicing silencer (ESS16) that interacts with hnRNP A/B in heterologous cell context. We here show that ESS16 promotes the recruitment of a protein complex containing hnRNP A1 and a 79-kDa protein in nuclear extracts from either proliferative erythroleukemia cells or cells induced to terminal differentiation. By using 2D gel fractionation and mass spectrometry, we unambiguously identified KSRP as the 79-kDa component interacting with ESS16. Furthermore, we show that KSRP slightly decreases in erythroleukemia cells induced to terminal erythroid differentiation. Yet, KSRP inducible knockdown, through stable transfection of small hairpin KSRP RNA, did not alter exon 16 splicing, suggesting that KSRP alone does not modulate the splicing event. Interestingly, absence of hnRNP A1 prevented KSRP from binding to ESS16. Reciprocally, KSRP interaction with ESS16 was recovered when hnRNP A1 expression is restored in hnRNP A1-null cells. Collectively, this study establishes that hnRNPA1 is part of a KSRP-containing RNP complex, and emphasizes that, aside from its function in AU-rich element-mediated mRNA decay and its role in microRNA biogenesis, KSRP associates with hnRNP A1 to bind an ESS. These findings further support the role of members of the KH-domain protein family in organizing large RNA-protein complex formation, rather than primarily in modulating specific splicing events.