Basal cell carcinoma of the skin is a common cancer that is associated with increased activity of the Sonic Hedgehog pathway. GLI proteins are the terminal effector molecules that interpret Hedgehog signals and overexpression of GLI1 and GLI2 in model organisms has been shown to give rise to neoplasia. The GLI1 gene is subject to several regulatory mechanisms that maintain appropriate expression including transcriptional, post-transcriptional, and post-translational controls. For example, posttranscriptional regulation generates alternative GLI1 isoforms with different 5' untranslated regions which exhibit differential translational activities.
Alternative splicing is a widespread phenomenon whereby multiple mRNA isoforms can be generated from a single genetic unit. Splicing decisions are influenced by proteins belonging to the serine/arginine (SR)-rich protein and the heterogeneous nuclear ribonucleoprotein (hnRNP) families, which bind to enhancer and silencer sequences, respectively. Additionally, recent studies have demonstrated a link between gene transcription and alternative processing outcomes. It was the aim of this project to discern regulatory mechanisms underlying control of exon skipping within human and mouse Gli1.
Co-transfection of minigene and splicing factor constructs was employed in detecting Gli1 processing. Stable and transient transfection into various cell lines such as HeLa, HaCaT keratinocytes, HEK293, and CHO revealed cell type specific differences in Gli1 regulation. Of the splicing factors tested, human Transformer2β1 (hTRA2β1), Polypyrimidine tract binding protein (PTB), SRp20, SRp40, and hnRNP A1 altered expression of Gli1 minigene 5' UTR isoforms in various cellular contexts.
PTB and hTRA2β1 resulted in altered expression of both human and mouse Gli1 minigenes. Putative PTB binding sites were identified surrounding the alternative exons and co-transfection of this factor resulted in exon skipping. hTRA2β1 simultaneously up-regulated inclusion of one mouse alternative exon as well as skipping of another alternative exon. Interestingly, hTRA2β1 co-transfection with the human GLI1 minigene increased levels of both human GLI1 isoforms.
Minigene promoter swapping, as well as comparison to the endogenous GLI1 transcripts, revealed dependence of pre-mRNA processing on transcriptional strength. A strong promoter (CMV) lead to greatly increased levels of ex on skipping and the weakest promoter (endogenous PGLI1) lead to most exon inclusion. However, no promoter specific sensitivities were observed for the splicing factors in the cell line tested.
Single Nucleotide Polymorphisms (SNPs) residing in human GLI1 exon 1 A were tested for functional relevance. Abrogation of putative splicing factor recognition sites was determined bioinformatically, and tested using co-transfection strategies. Three splicing factors tested - SRp40, TRA2β1 and ASF - exhibited differential activities with respect to sequence variants. Translational efficiency of GLI1 isoforms and regulation by nonsense-mediated decay were also examined and revealed other potential GLI1 regulatory mechanisms.
This study provides preliminary information regarding Gli1 alternative processing. The findings will contribute to the expanding field of post-trancriptional regulation, as well as to the regulation of an important mediator of Sonic Hedgehog signalling in BCC tumourigenesis.