The balance of splicing : A novel insight into the splicing regulation of high-risk HPV E6 and E7 oncogenes

Abstract: HPV is associated with several cancers. The genome consists of a long control region, early (E1, E2, E4, E5, E6 and E7) and late (L1 and L2) genes. The E6 and E7 proteins prevent cells from entering apoptosis and regulate the cell cycle. A deregulated expression of these can result in malignant transformations. Therefore, a deeper understanding of their regulation is important. HPV gene expression regulation occurs mainly through alternative pre-mRNA splicing with many splice events being mutually exclusive. This is the case with E6 and E7. E6 is expressed from the intron-retained mRNA while E7 is expressed from a spliced mRNA. In this thesis it was aimed to understand the alternative splice events of HPV16 E6 and E7 mRNAs by identifying regulatory elements controlling these splice events. A strong enhancer, downstream of SA409, was identified. It consists of a perfect bipartite repeat and mutations in the element disrupts SA409 splicing. Trans-acting factors were determined to TRAP150 and BCLAF1 (Paper I). Downstream of this is another cis-element. It consists of a GGGG-motif with a silencing effect on SA409 splicing. Two additional cis-elements, one at the end of the E6 ORF and the other at the start of the E7 ORF, were additionally found. These are suggested to hybridize forming an internal-loop structure when analyzed in silico. The cis-element at the end of the E6 ORF is context dependent, functioning as an enhancer or silencer depending on if the E7 cis-element is included or not. It was identified as ATCATCA (Paper III). The cis-element at the start of the E7 ORF was a silencer and consisted of an AUG-rich element. Two trans-acting factors interacted with it, hnRNP A1 and hnRNP A2, and prevented SA409 splicing. However, hnRNP A1 increased the intron-retained E6 mRNA, while hnRNP A2 redirected splicing to a downstream acceptor, SA742 (Paper IV). SA742 was additionally found to be regulated by another cis-element, within the E1 ORF. It consisted of three GGG/GGGG motifs and the integrity of these and of SD880 were important for SA742 usage, indicating the importance of regulatory factors in the modulation of the splicing modes: intron definition and exon definition. The trans-acting factor was hnRNP H (Paper II). In this thesis five cis-elements and five trans-acting factors affecting splicing regulation within the E6 and E7 ORFs were identified. With this knowledge to build upon several important targets to change or disrupt splicing were identified.