Gene expression is regulated through a dynamic interconnected web of systems with its rich complexity only further appreciated following the Human Genome Project. The genome by itself not containing all information on a biological state has led to growing interest in the regulation of its expression, including through chemical modification deposited within mRNA transcripts. Here, I explore the impact of the two most abundant mRNA modifications, N6-methyladenosine (m6A) and pseudouridine (Ψ) on gene expression by study of modification writer and reader enzymes, which respectively deposit and interact with transcript modifications. In Chapter 2, I show how m6A reader protein hnRNPG can accommodate mutual, co-transcriptional interactions with RNA Polymerase II and m6A-modified RNA through development of a spin-down assay for low-complexity protein interactions. In Chapter 3, I demonstrate that type I reader protein YTHDC1 interacts with RNA Polymerase II through its structured YTH-domain, which is outcompeted by its canonical ligand in m6A. In Chapter 4, I perform a bioinformatic survey on the connection between m6A writers and readers on tRNA. In Chapter 5, I detail our newly developed NanoSPA platform for direct, simultaneous long-read sequencing of m6A and Ψ along the same mRNA transcript. Using this platform, we demonstrate crosstalk between m6A and Ψ on modification concurrence and translational efficiency. Together, the work here expands the mechanistic understanding of mRNA modification writers and readers in regulation of gene expression.