Eukaryotic gene expression is an essential process for proper cell differentiation, development, and the cell’s response to environmental signals. Pre-mRNA splicing is an important part of the eukaryotic gene expression program. Splicing contributes to protein diversity, gene expression regulation, evolution, and genetic diseases. Understanding the intricacies of pre-mRNA splicing is important for understanding gene expression and for the rational design and development of new therapies for genetic diseases.The regulation of pre-mRNA splicing is a highly combinatorial process that relies on many cis- and trans-acting elements. Some of these elements include splice site strength and the intron-exon architecture. It is proposed that spliceosome assembly can either occur across the intron (referred to as intron definition) and across the exon (referred to as exon definition). Selecting between these modes of spliceosome assembly is thought to be dictated by intron architecture. Other studies have demonstrated that the proximity between the 5' splice site and its intronic 3' splice site plays a critical role in splice site selection. In chapter 2 we conducted a genome-wide computational analyses to evaluate the proximity rules in the context of intron and exon definition. Our computational studies were complemented using designer mini-genes in cell transfection assays to evaluate the impact of splice site proximity in alternative splicing. The ability to regulate gene expression allows the cell to adjust to its ever-changing needs and external cues. Aberrant regulation of gene expression is linked to diseases such as cancer. One major contributor to modulate gene expression is through the regulation of mRNA stability. A change in mRNA stability can lead to differing protein expression levels while alternative splicing primarily promotes protein diversity. The work described in Chapter 3 outlines what gene features impact mRNA stability and how alternative splicing can influence mRNA stability. Data was generated by conducting a 24-hour 4sU pulse-chase RNA-seq experiment. Our computational analyses allowed us to explore the relationship between mRNA stability and gene and/or exon length. In addition, we established a pipeline to derive exon and mRNA isoform half-lives to investigate the influence of alternative splicing on mRNA stability. Cancer cells have been known to have unique metabolic needs for proliferation. One such need is the cancer cell’s metabolic addiction to methionine, referred to as the “Hoffman effect.” While the Hoffman effect has been observed in a wide array of cancer cells, the mechanisms by which it arises, and controls tumorigenesis are not fully understood. In chapter 4, gene expression analyses of methionine-dependent and independent cell lines reveal that splicing dysregulation is linked to methionine dependence. In particular, proper methylation of a general spliceosomal component is implicated as link between changes in splicing fidelity and the accessibility of exogenous methionine in cancer cells.