Investigating the contribution of alternative polyadenylation to the regulation of gene expression

Thesis

Around 70% of human genes have been found to contain multiple cleavage and polyadenylation (pA) sites. The alternative usage of these different pA sites is referred to as alternative cleavage and polyadenylation (APA). A wide range of studies have described switches in APA profiles when comparing different cell types and different cell states, notably general shifts to shorter APA isoforms upon the onset of cancer. These shifts have been described at both the individual gene and transcriptome levels. However, the extent to which these changes in APA profiles are contributing to changes in gene expression, and thereby altering the observed phenotype, is still under debate.

This thesis develops a novel subcellular fractionation approach to isolate those genes where the alternative use of different pA sites results in APA isoforms that differ in either the cytoplasmic stability or nuclear export rates. These differences would materialise as a differential representation of the resulting APA isoforms in the cytoplasmic and nuclear RNA fractions. This approach was applied to 12 human cell lines using a combination of publicly available generic poly-A+ RNA-sequencing data and 3'end sequencing performed here. Eleven out of twelve of these cell lines showed a significant bias towards an overrepresentation of the shorter, proximal APA isoform in the cytoplasm, which fits with the general model that 3’untranslated regions (3'UTRs) are mainly repressive and promote cytoplasmic instability. Comparisons of the isolated genes for each cell line show that differential regulation of APA isoforms tends to be largely cell type specific.

Application of this subcellular fractionation approach to a cell-line based model for colorectal cancer progression highlights a small cohort of genes where APA is likely to have an impact on gene expression, potentially contributing to colorectal cancer progression. This analysis provides an ideal starting point to study these APA events at the individual gene level to elucidate fully whether APA is having a significant impact on gene expression.

Furthermore, post-transcriptional regulatory pathways governing these differential regulation events were investigated, including the contribution of micro-RNAs (miRNAs) to the differential regulation of APA isoforms. Unexpectedly, depletion of the endoribonuclease III, DICER1, reveals that it does not only shape the steady-state cytoplasmic APA profile through its well-known function in miRNA biogenesis, but also influences pA site choice in the nucleus.

Authors: Neve, J

• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford