Student Theses and Dissertations

Date of Award

2021

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Darnell Robert Laboratory

Abstract

Advancements in DNA sequencing technology and the implementation of clinical genome and exome sequencing have allowed for the identification of candidate variants and genes essential in brain development and pathogenesis of neurological diseases, yet there is still much to be learned. It is estimated that the molecular diagnostic rate of whole exome sequencing (WES) of constitutional diseases is between 9-41%; however, it is thought that diagnostic yield could be much improved by gaining a better understanding of individual variation and regulation at the transcript level. Identifying the root cause of diversity between humans and specifically in the human brain has been a long-standing question for the scientific field. At the turn of the 20thcentury, Santiago Ramón y Cajal illustrated thousands of neurons spanning across brain regions to irrefutably demonstrate the morphological complexity and diversity of individual neurons (Levine and Marcillo, 2008). Discoveries spanning centuries continue to drive investigators to question not only why neuronal diversity exists, but how a single genome gives rise to the remarkable heterogeneity observed between single cells, tissue structures, and circuit connections in the brain. Post-transcriptional gene regulation contributes to organism complexity in eukaryotes. RNA regulation is strictly controlled by RNA-binding proteins that are important for the coordination and regulation of gene expression and eukaryotic cells have evolved intricate posttranscriptional mechanisms that permit for precise spatial and temporal control of RNA steady state levels in any given cell. Alternative RNA processing via RNA binding proteins underlies distinct phenotypic and functional diversity specifically observed among mammalian cells. Alternative splicing (AS) and alternative polyadenylation (APA) are widely utilized to expand and diversify both transcriptome and proteome. Alternative RNA processing allows one gene to produce multiple transcripts with distinct coding and regulatory sequences giving rise to multifaceted protein function. RNA binding proteins dictate alternative RNA processing which is a key player in most, if not all, biological processes. Understanding how RNA binding proteins regulate RNA steady state levels and alternative RNA processing requires knowledge of the genomic positions to which these proteins function in vivo. Mouse models have facilitated genome-wide, unbiased discovery of RNA regulatory sites, and the link between causation and functional effect with extraordinary resolution and specificity. The inability to translate many RNA regulatory events from the mouse to the human genome emphasizes the need to perform biochemistry on the human brain directly to compare with the mouse. Identification of RNA regulatory regions via CLIP-sequencing provides valuable information as to how human RNA profiles are dictated and controlled in normal and disease states. Studying neuronal splicing factor NOVA elucidates previously unidentified non-coding RNA regulatory sites as functional and unique to human neurons. We show that human expansion in NOVA regulatory elements diversifies the RNA landscape specifically in human neurons and differentially across brain regions. Taken together, these findings demonstrate mouse to human conserved relationships between RNA binding proteins and targets, however the activity, specificity, and usage of RNA regulatory elements is largely species-specific.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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