Ogunsola, Samuel2026-09-032026-09-032026-08-242026-08-262026-09-03http://hdl.handle.net/1993/40061The mammalian transcriptome is shaped by interconnected layers of co- and post-transcriptional regulation, yet the mechanisms that maintain its integrity remain incompletely understood. This thesis investigates two dimensions of transcriptome integrity regulation: the genome-wide role of an evolutionarily conserved mammalian genomic signature in regulating alternative splicing, and the capacity of a DNA methyltransferase inhibitor to restore transcript length in cancer cells. Guanosine-tract closely upstream of an AG dinucleotide (G-tract-AG signatures) emerged in mammals and they have been found in over 2000 annotated human 3′SS as a repressor of 3′AG usage through hnRNP H1/F binding. However, their distribution and function across the broader non-coding genome were unknown. Here, we demonstrate that G-tract-AG signatures are widely distributed across mammalian non-coding regions, occurring over six million times in the human genome. Approximately 9,000 G-tracts in G-tract-AG signatures within human genes are disrupted by single nucleotide variants (SNVs) of the cis-splicing quantitative trait loci in the Genotype-Tissue Expression project. Mechanistically, G-tract elements repress splicing in vitro primarily by stalling the second transesterification step. Moreover, we also showed that it stabilized a repressive stem-loop structure between the branchpoint and the adjacent AG. Interestingly, G-tract-disrupting SNVs destabilize this stem-loop, relieve AG repression, and activate cryptic splicing, generating novel transcript isoforms. These G-tract-disrupting SNVs include thousands of rare alleles implicated in Mendelian disease, providing a mechanistic framework for functional annotation of non-coding variants. At the polyadenylation level, treatment of GH3 pituitary tumor and MOLM-13 leukemia cells with the DNA methyltransferase inhibitor 5-aza-cytidine promotes a transcriptome-wide shift from proximal polyadenylation sites toward genomic terminal exons with more optimal poly(A) signal motifs. This shift is accompanied by upregulation of anti-termination factors, SCAF4 and SCAF8, downregulation of the early termination enhancer, E2F2. These findings reveal a previously unrecognized role for 5-aza-cytidine in driving a switch from shortened to full-length mRNA transcripts in tumor cells. Taken together, this thesis establishes that transcriptome integrity and diversity emerge from the interplay of multiple mechanisms, and that perturbation of any of these has broad consequences in health and disease.engAlternative SplicingGenetic VariantsSingle Nucleotide VariantsRNA SequencingmRNA Splicing