Different mechanisms contribute to regulate the expression of Disc Large 1 Oncosuppressor, a target of high-risk HPV E6 proteins

ANA LAURA CAVATORTA, MARINA BUGNON VALDANO, FEDERICO MARZIALI, FLORENCIA FACCIUTO, LAWRENCE BANKS AND DANIELA GARDIOL.

Trieste

Congreso; DNA Tumour Virus Meeting; 2011

ICGEB

The Disc Large 1 oncosuppressor (DLG1) is involved in cell polarity control and tissue architecture maintenance and was shown to be targeted by HPV E6 oncoproteins and to be down-regulated during malignant progression in human carcinomas. However, the mechanisms controlling DLG1 expression are poorly understood. We have previously reported that Snail transcription factors, up-regulated in a number of tumors, are involved in DLG1 down-regulation. Bioinformatic analysis of DLG1 promoter showed consensus binding sites for others factors such as Sp1, which plays an important role in cell growth and differentiation. By luciferase assays and immunofluorescence we demonstrated that Sp1 is involved in DLG1 up-regulation. Futhermore, we showed by qRT-PCR that growth factors, involved in tumour-progression, reduce DLG1 transcription in a dose-dependent manner. To further examine DLG1 regulation, we analyzed the 5’ends of DLG1 transcripts by rapid amplification of cDNA ends-PCR. We identified an alternative splicing event in the 5’region of DLG1 mRNA that generates transcripts with two different 5’UTRs. We showed by reporter assays that the DLG1 5’UTR containing an extra exon interferes with the translation of a downstream ORF. However, no significant differences in mRNA stability among the DLG1 5’UTR variants were observed. Sequence analysis of the additional exon showed the presence of an upstream short ORF (uORF) which is lost in the short version of the 5’UTR DLG1. By mutagenesis and luciferase assays we analyzed the contribution of this uORF in reducing translation efficiency, and demonstrated that its disruption can revert to some extent the negative regulation of the large 5’UTR. Using computational modeling we showed that the large DLG1 5’UTR isoform forms a more stable structure than the short version, and this might contribute to its ability to repress translation. This study shows for the first time that differential expression of alternatively spliced 5’UTRs could result in changes in DLG1 abundance.