title: Regulation of human PTCH1b expression by different 5' untranslated region cis-regulatory elements.
abstract: 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.
authors.affiliation: a Laboratory for Hereditary Cancer ; Division of Molecular Medicine ; Ruđer Bošković Institute ; Zagreb , Croatia.
authors.forename: Petar
authors.initials: P
authors.lastname: Ozretić
authors.forename: Alessandra
authors.initials: A
authors.lastname: Bisio
authors.forename: Vesna
authors.initials: V
authors.lastname: Musani
authors.forename: Diana
authors.initials: D
authors.lastname: Trnski
authors.forename: Maja
authors.initials: M
authors.lastname: Sabol
authors.forename: Sonja
authors.initials: S
authors.lastname: Levanat
authors.forename: Alberto
authors.initials: A
authors.lastname: Inga
mesh_terms: D020121:5' Untranslated Regions; D001483:Base Sequence; D015687:Cell Hypoxia / Q000235:genetics; D005786:Gene Expression Regulation; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D000067556:Internal Ribosome Entry Sites; D061986:MCF-7 Cells; D008969:Molecular Sequence Data; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D020121:5' Untranslated Regions; D000067556:Internal Ribosome Entry Sites; C000606236:PTCH1 protein, human; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D020033:Protein Isoforms; D011956:Receptors, Cell Surface
keywords: 5'UTR; 5′UTR, 5′ untranslated region; CGG repeats; Fluc, Firefly luciferase; Hedgehog-Gli; Hh-Gli, Hedgehog-Gli; IRES; IRES, internal ribosome entry site; POL, polysome-associated; PTC1-L, protein patched homolog 1; PTCH1; Rluc, Renilla luciferase; SUB, subpolysomal; isoform L PTCH1b, Patched 1 gene, transcript variant 1b; uAUG; uAUG, upstream AUG codon; uORF; uORF, upstream open reading frame
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