Regulation of human PTCH1b expression by different 5' untranslated region cis-regulatory elements.
RNA Biol
; 12(3): 290-304, 2015.
Article
em En
| MEDLINE
| ID: mdl-25826662
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.
Palavras-chave
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
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Biossíntese de Proteínas
/
Regulação da Expressão Gênica
/
Receptores de Superfície Celular
/
Regiões 5' não Traduzidas
/
Sítios Internos de Entrada Ribossomal
Limite:
Humans
Idioma:
En
Revista:
RNA Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2015
Tipo de documento:
Article
País de afiliação:
Croácia