Naslov
Functional Analysis of Cis-Regulatory Elements from 5' Untranslated Region of PTCH1b Gene

Autori
Ozretić, Petar ; Bisio, Alessandra ; Musani, Vesna ; Trnski, Diana ; Sabol, Maja ; Levanat, Sonja ; Alberto, Inga

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Book of Abstracts of of the Congress of the Croatian Society of Biochemistry and Molecular Biology „The Interplay of Biomolecules“, HDBMB2014 / Katalinić, Maja : Kovarik, Zrinka - Zagreb : Hrvatsko društvo za biokemiju i molekularnu biologiju (HDBMB), 2014, 65-65

ISBN
978-953-955551-5-1

Skup
Congress of the Croatian Society of Biochemistry and Molecular Biology „The Interplay of Biomolecules“, HDBMB2014

Mjesto i datum
Zadar, Hrvatska, 24.09.2014. - 27.09.2014

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Domaća recenzija

Ključne riječi
PTCH1b; 5'UTR; CGG REPEATS; uORF; uAUG; IRES

Sažetak
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.

Izvorni jezik
Engleski

Znanstvena područja
Biologija, Temeljne medicinske znanosti

POVEZANOST RADA