Project Summary
Dysregulation of RNA-binding protein (RBP)-RNA interactions impacts the coordination of RNA fate with
severe implications for human health. RBPs interact directly with RNA processing machinery to modulate RNA
localization, degradation, and translation independently of transcriptional regulation. Disruptions in RBPs or
their RNA binding sites can lead to fluctuations in oncogene or tumor-suppressor gene expression and
malignant changes in cellular homeostasis. Understanding the molecular functions of RBPs in regulating gene
expression is then imperative for predicting metastatic progression in cancers with RBP mutant gene
signatures. Recent work has led to the discovery of thousands of human RBPs, but much less has been done
to systematically describe the function that each protein and its individual domains have on regulating their
RNA partners. Additionally, the functional organization of RBPs beyond their distinct and generally
well-characterized RNA-binding domains (RBDs) is not fully understood. Until recently, it was assumed that
RBPs were effectively non-modular outside of RBDs; however, current studies suggest that RBPs may be
composed of separable ‘effector’ domains that interact with processing machinery and promote regulatory
activity. I hypothesize that most RBPs contain distinct effector domains that post-transcriptionally regulate
protein abundance through interactions with cellular machinery that controls RNA decay or translation.
The Bintu lab has developed a high-throughput recruitment assay to recruit 80 amino acid protein portions, or
tiles, to a synthetic DNA reporter gene. I propose to extend this methodology to identify functional effector
domains within human RBPs, where they also may be more sensitive to oncogenic mutation. First, I will use a
high-throughput assay I recently developed to recruit tens of thousands of protein tiles to a synthetic reporter
RNA, evaluate their effects on downstream protein expression, and determine the mechanism by which they
regulate RNA levels. This will allow me to identify annotated oncogenic mutations that overlap effector domains
and likely cause disease by disrupting RBP regulatory activity. Second, it is currently difficult to predict how
loss of RBP activity affects a single bound transcript, largely because RBPs bind at different positions and in
different copy numbers on each of their multiple partner RNAs. I will quantitatively compare how variation in
RBP and effector domain positioning and occupancy along transcripts affects gene expression and eventual
different cancer phenotypes. Finally, I will conduct a mechanistic investigation into METTL3, a known
RNA-modifying enzyme whose overexpression is believed to drive the development of multiple cancers. I will
determine its RNA regulatory potential independent of modification writing and query the direct correlation
between RNA modifications and overall expression. This work will be the first to systematically identify
functional domains in RBPs and will increase understanding of how dysregulation of RBP function leads to
cancer through altered interactions with crucial cellular machinery.