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PTCH1 gene codes for a 12-pass transmembrane receptor with a negative regulatory role in the Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, a disorder characterized by developmental abnormalities and tumor susceptibility. The autosomal dominant inheritance, and the evidence for PTCH1 haploinsufficiency, suggests that fine-tuning systems of protein patched homolog 1 (PTC1) levels exist to properly regulate the pathway. Given the role of 5' untranslated region (5'UTR) in protein expression, our aim was to thoroughly explore cis-regulatory elements in the 5'UTR of PTCH1 transcript 1b. The (CGG)n polymorphism was the main potential regulatory element studied so far but with inconsistent results and no clear association between repeat number and disease risk. Using luciferase reporter constructs in human cell lines here we show that the number of CGG repeats has no strong impact on gene expression, both at mRNA and protein levels. We observed variability in the length of 5'UTR and changes in abundance of the associated transcripts after pathway activation. We show that upstream AUG codons (uAUGs) present only in longer 5'UTRs could negatively regulate the amount of PTC1 isoform L (PTC1-L). The existence of an internal ribosome entry site (IRES) observed using different approaches and mapped in the region comprising the CGG repeats, would counteract the effect of the uAUGs and enable synthesis of PTC1-L under stressful conditions, such as during hypoxia. Higher relative translation efficiency of PTCH1b mRNA in HEK 293T cultured hypoxia was observed by polysomal profiling and Western blot analyses. All our results point to an exceptionally complex and so far unexplored role of 5'UTR PTCH1b cis-element features in the regulation of the Hedgehog-Gli signaling pathway.

Background PTCH1 tumor suppressor gene encodes for a 12-pass transmembrane receptor with a negative regulatory role in Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome, disorder characterized by developmental abnormalities and tumor susceptibility. Most of the malformations are caused by PTCH1 haploinsufficiency, indicative of fine-tuning needed to properly regulate the activity of pathway which is involved in pathogenesis of various tumors. Since in our previous study we identified 5 to 8 CGG repeats located 4 bases upstream of a translation initiation site, our aim in this study was to examine how 5’ untranslated region (5’UTR) regulates the expression of PTCH1 transcript 1b. Material and methods All potential cis-regulatory elements in PTCH1b 5’UTR were studied by various in silico tools and gene reporter assays. We tested the influence of 5’UTR length and CGG-repeat polymorphism in a way that in pGL3-P plasmid we cloned either 188- or 300bp-long 5’UTR, each harboring 5 to 8 CGG repeats, upstream of firefly luciferase gene. Site-directed mutation (SDM) was used to test the significance of predicted upstream open reading frames (uORF). We also constructed bicistronic pRuF vectors by cloning each PTCH1b 5’UTR between Renilla and firefly luciferase gene, since the last 76bp in both 5’UTR were predicted as an internal ribosome entry site (IRES). Dual luciferase assays and qPCR were performed in MCF-7, HCT 116 and HEK 293T cells. Results Reporter assays showed that shorter 5’UTR significantly increased reporter activity, with a subtle reduction with higher number of repeats. Longer 5’UTR led to much reduced reporter activity, without a difference among repeats. Luciferase mRNA quantification showed that both 5’UTR lengths significantly increased transcription. SDM proved hypothesis that 2 potential uORF, contained only in the first 112bp of 300bp-long 5’UTR, might account for this severe reduction in reporter activity. Both 5’UTR lengths significantly increased firefly luciferase activity of pRuF vectors (proved by PCR this is not due to alternative splicing), with no difference among repeats. Firefly luciferase activity was significantly reduced when predicted IRES motif was removed from pRuF plasmid. Firefly/Renilla luciferase mRNA ratios were the same as for empty vector, indicating that observed higher firefly luciferase activity should be due to a post-transcriptional regulation, i.e., cap-independent translation of firefly luciferase mRNA. Conclusions All our results point to the exceptionally complex and so far unexplored role of 5’UTR in the regulation of PTCH1b expression, whilst the existence of an IRES motif would enable Ptch1 protein to be synthesized under conditions when the general level of protein synthesis is reduced, such as in hypoxia, which is known activator of Hedgehog-Gli pathway.

PTCH1 tumor suppressor gene encodes for a transmembrane receptor with a negative regulatory role in Hedgehog-Gli signaling pathway. PTCH1 germline mutations cause Gorlin syndrome which is characterized by developmental abnormalities and tumor susceptibility. Most of malformations are caused by PTCH1 haploinsufficiency, indicative of fine-tuning needed to properly regulate pathway activity. After identifying 5 to 8 CGG repeats close to translation initiation site, our aim was to examine how 5’ untranslated region (5’UTR) regulates the expression of PTCH1 transcript 1b. All potential PTCH1b 5’UTR cis-regulatory elements were studied by various in silico tools and gene reporter assays. We tested the influence of 5’UTR length and CGG-repeat polymorphism by cloning either 188- or 300bp-long 5’UTR, each harboring 5 to 8 CGG repeats, in pGL3-P plasmid upstream of firefly luciferase gene (Fluc). Site-directed mutagenesis (SDM) was used to test the significance of predicted upstream open reading frames (uORF). Bicistronic pRuF vectors were constructed by cloning PTCH1b 5’UTRs between Renilla and firefly luciferase genes, since the last 76bp in PTCH1b 5’UTR were predicted as an internal ribosome entry site (IRES). Dual luciferase assays and qPCR were performed in MCF-7, HCT 116 and HEK 293T cells. Reporter assays showed that shorter 5’UTR significantly increased reporter activity, with a subtle reduction with higher repeat number. Longer 5’UTR led to much reduced reporter activity, without a difference among repeats. Fluc mRNA levels showed that both 5’UTRs significantly increased transcription. SDM proved hypothesis that 2 potential uORFs, present only in 300bp-long 5’UTR, might account for this severe reduction in Fluc activity. Both 5’UTRs significantly increased Fluc activity of pRuF vectors (proved by PCR this is not due to alternative splicing), with no difference among repeats, while Fluc activity was significantly reduced after removing predicted IRES motif. Firefly/Renilla luciferase mRNA ratios were the same as for empty vector, indicating that observed higher Fluc activity should be due to a post-transcriptional regulation, i.e., cap-independent translation of Fluc mRNA. All our results point to exceptionally complex and so far unexplored role of 5’UTR in the regulation of PTCH1b expression, whilst the existence of an IRES would enable PTC1 protein to be synthesized under conditions when general level of protein synthesis is reduced, such as in hypoxia.