Messenger RNAs (mRNAs) have to undergo a series of post-transcriptional processing steps before translation. One of the post-transcriptional steps - 3' end processing, which consists of cleavage and polyadenylation, is critical for delimiting the 3' end of mRNA and determining regulatory elements for downstream post-transcriptional/translational regulation. Like another well-characterized mRNA processing step - splicing, 3' end processing is very flexible due to the diversity of trans-acting factors and cis-acting elements in the 3' end of mRNA. In recent years, the differential usage of alternative polyA sites (APA) of the same gene, which leads to mRNA isoforms of different 3' UTR, has been increasingly revealed by both experimental and computational studies. More significantly, the global changes of 3' UTR length have been observed in multiple clinical settings, particularly in the cancer cells. However, the depiction of APA phenomenon does not synchronize the efforts to study the mechanism underlying APA biogenesis.
In this thesis, we first describe general principle and pipeline to identify APA in different biological or clinical conditions using various high throughput sequencing techniques. After that, we present the work about the global impacts of two RNA binding proteins (ESRP/aCP) and one core 3' end processing factor (CstF64 and its paralog CstF64τ) on the regulation of APA. The APA identification analyses and motif analyses suggest a wide range of APA associated with the expression change of those proteins in different cell lines. In addition, for each protein, we have collect substantial evidence about the mechanism underlying the APA induction. Our findings could provide significant insights into the APA regulation mechanisms.
In addition, we also conducted a research on the induction of APA in JEG-3 cells as a response to the change of oxygen supply (Hypoxia and Normoxia). Using a robustness protocol for specifically sequencing 3' end of mRNA, we identified more than 500 APA events and revealed a global shortening pattern of 3' UTR length as a result of hypoxia.
The work on APA in this thesis largely increases the understanding of APA regulation by various proteins and provided new evidence for the APA in clinical condition.