Student: Peter Freese

Lab: Burge

Title: "Biochemical and Functional Characterization of Human RNA Binding Proteins"

RNA not only shuttles information between DNA and proteins but also carries out many other essential cellular functions controlled by approximately one thousand RNA binding proteins (RBPs). In this thesis, I describe the affinity landscapes of the largest set of human RBPs to date elucidated through a high-throughput version of RNA Bind-N-Seq (RBNS), an unbiased in vitro assay that determines the primary sequence, secondary structure, and contextual preferences of an RBP. The 78 RBPs bound an unexpectedly low diversity of RNA motifs, implying convergence of binding specificity toward a small set of RNA motifs characterized by low compositional complexity. Offsetting the low diversity of sequence motifs, extensive preferences for contextual features beyond short linear motifs were observed, including bipartite motifs, flanking nucleotide content, and preference for or against RNA structure. These features likely refine which motif occurrences are selected in cells, enabling RBPs that bind the same linear motif to act on distinct subsets of transcripts. Additionally, RBNS data is integrated with complementary in vivo binding sites from eCLIP-seq and functional (RNAi/RNA-seq) data produced through collaborative efforts with the ENCODE consortium. These data enable creation of “RNA maps” of RBP activity in pre-mRNA splicing and gene expression levels. Together, these novel data augment the catalog of functional elements encoded in the human genome to include those that act at the RNA level and provide a basis for how RBPs select their RNA targets, a fundamental requirement in more fully understanding RNA processing mechanisms and outcomes.