SUMMARY Protein synthesis is primarily regulated at the initiation stage, allowing rapid and reversible control of gene expression. Whereas cellular mRNAs require a 5′ cap to initiate translation, many viral mRNAs require an internal ribosome entry site (IRES). IRESs fold into three-dimensional structures and initiate translation through non- canonical interactions with cellular translation initiation machinery. Despite decades of research, many important questions remain unanswered about how IRESs initiate translation, particularly for the structurally complex Type I and Type II IRESs. Using biochemical studies, we have observed previously unknown IRES RNA structural changes during translation initiation. We have also leveraged powerful cryo-EM techniques to obtain high- resolution Type II IRES structures at the final stage of initiation. In the first theme of this proposal, we will build upon these results and integrate biochemical, chemical, molecular, and structural techniques to determine how IRES structural dynamics regulate translation initiation of Type I and Type II IRESs. Our long-term goal is to combine these mechanistic insights with high-throughput RNA structural probing and translation complex profiling to understand how natural IRES mutations affect structural dynamics, translation efficiency, and virulence. Type I and Type II IRESs are crucial for circular mRNA (circRNA) technology, which has gained interest as a new mRNA therapeutic platform. Current circRNA design will benefit from incorporating modified nucleosides, such as pseudouridine (ψ) or N1-methylpseudouridine (m1ψ), that are critical for reducing the immunogenicity of linear mRNAs. However, ψ/m1ψ modification inactivates Group I introns and Type I/II IRESs— key RNA elements for circularizing and translating circRNAs, respectively. This result is paradoxical given that uridine, ψ, and m1ψ are virtually interchangeable in transcription and translation due to their structural similarities. Our overall hypothesis is that while these modifications maintain and stabilize the Watson-Crick base pairing, they disrupt certain non-canonical base pairing or tertiary interactions critical for folding large non-coding RNAs. The goal of the second theme of our research is to solve this paradox and determine how ψ and m1ψ affect RNA structures and translation. Using the Group I intron and Type II IRES as our model molecular systems, we will use RNA structural probing, biochemical, and molecular techniques to elucidate how these modified nucleotides affect RNA tertiary interactions and folding beyond canonical Watson-Crick base pairing. Our results will provide fundamental knowledge of how base modifications affect RNA folding and function, informing the development of potent mRNA therapeutics.