Gene expression is controlled by a complex and interconnected regulatory system. In this thesis, I explore how RNA modifications impact protein binding, co-transcriptional regulation, and sequencing signatures. The cellular functions of the abundant messenger RNA (mRNA) modification N6-methyladenosine (m6A) are mediated by m6A reader proteins. Some m6A reader proteins selectively bind m6A-modified RNAs by recognizing motifs that become accessible upon an m6A-induced change in RNA structure. In Chapter 2, I use biophysical methods to examine how m6A modification alters the structure of an RNA hairpin to enhance binding of the protein heterogeneous nuclear ribonucleoprotein C (hnRNPC). In Chapter 3, I describe the discovery of another m6A reader protein, hnRNPG, which binds to a motif that becomes exposed upon m6A modification of an RNA hairpin. Unlike hnRNPC, hnRNPG binds to a motif that includes the m6A site. Moreover, hnRNPG uses Arg-Gly-Gly (RGG) repeats in a low-complexity region to selectively bind to m6A-modified RNA. In Chapter 4, I explore the cellular functions of hnRNPG binding to m6A-modified RNAs. The hnRNPG protein binds to the phosphorylated C-terminal domain of RNA polymerase II and to nascent m6A-modified RNA for co-transcriptional, m6A-dependent gene regulation. In Chapter 5, I build on existing sequencing methods for the detection of another RNA modification, pseudouridine (Ψ). This work spans a variety of perspectives on the impact of RNA modifications, from a biophysical study of the effect of m6A on RNA structure and protein binding, to an investigation of the cellular functions of m6A in gene regulation, to method development for the detection of pseudouridines in high-throughput sequencing data. Together, these diverse perspectives demonstrate the widespread impact of RNA modifications.