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 N 6-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 (Psi). This work spans a variety of perspectives on the impact of RNA modifications, from a biophysical study of the effect of m6 A 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.