Abstract: Protein synthesis is an essential process that is highly regulated. In eukaryotes, protein synthesis is highly influenced by RNA sequences and structures in mRNA transcript leaders (5' UTRs). In particular, upstream open reading frames (uORFs) alter the rate of ribosome loading on mRNA coding regions by acting as decoys to scanning ribosomes. However, these elements can be difficult to identify, as the locations of mRNA transcript leaders are often poorly annotated. My talk will cover three vignettes of studies in our lab on transcript leader function. First, we mapped thousands of transcript leaders and uORFs in multiple yeast species, assayed their influence on gene expression, and built models that quantitate the roles of sequence and structural features on mRNA translation. Next, I will discuss how errors in mouse mRNA annotations led to incorrect models of transcript leader function, resulting in a substantial paradigm "unshift" in mammalian developmental biology. Finally, I will describe our current research, in which we apply the tools we developed in yeast and the lessons we learned from mice to study the functions of human transcript leaders using comparative genomics, RNA structure probing, and massively parallel reporter assays.