Comprehensive Identification and Characterization of Cajal Body Components

Date of Award

Spring 2022

Document Type


Degree Name

Doctor of Philosophy (PhD)


Molecular Biophysics and Biochemistry

First Advisor

Neugebauer, Karla


Each cell’s nucleus is a safe harbor for the genome (DNA), ensuring the integrity of all of the genes that contribute to development and health of an organism. What’s more, the 3-dimensional (3D) organization of the cell nucleus regulates how genes are expressed to give each cell its unique characteristics. In addition to chromosomes, the nucleus contains nuclear bodies, which are functionally distinct yet membraneless compartments that likely form through liquid-liquid phase separation (LLPS). Nuclear bodies concentrate specific proteins and RNAs on distinct chromosomal DNA regions to regulate gene expression. One of these nuclear bodies, the Cajal Body (CB), is implicated as a site of synthesis and assembly for components of the spliceosome, a molecular machine responsible for maturation of nearly every human messenger RNA (mRNA). CBs are necessary for normal development, since vertebrate embryos depleted of the CB scaffolding protein coilin do not properly process mRNA and cannot support growth. Remarkably, the composition of nuclear bodies is not well defined. This lack of knowledge is the most important current obstacle to understanding how nuclear bodies govern gene expression. This dissertation aims to discover all of the components of the CB systematically, and to determine which are necessary for CB assembly.I have adapted and applied state-of-the-art proximity biotinylation techniques (APEX2) to obtain a comprehensive list of CB proteins by mass spectrometry. I identified 70 new CB proteins, nearly doubling the number of known constituents. Of these, IRF2BP1 is the first DNA binding protein to be identified in CBs. I have performed a screen depleting each CB protein individually and analyzed changes to CB number and shape. As a result, I found that 46 CB proteins are necessary for proper CB assembly. The siRNA screen revealed three different phenotypes of improper CB assembly, 1) decreased number of CBs per nucleus, 2) increased number of CBs per nucleus and 3) relocalization of coilin to nucleoli. Further analysis of the increased number phenotype revealed components of the 60S large ribosomal subunit (RPL proteins) as regulators of CB assembly. This is the first study to demonstrate regulation of CBs by ribosomal proteins. Next, I characterized the increased coilin foci upon RPL knockdown and found effects to CB morphology and composition. CBs after RPL knockdown showed a reduction in snRNP proteins and lost their distinct substructure. Cajal bodies are normally made up of two subunits, a coilin containing domain and an SMN containing domain. Upon RPL KDs, this subdomain structure was lost, instead forming one domain with both coilin and SMN. Cajal bodies are known to form on actively transcribing snRNA and histone gene loci. I performed chromatin immunoprecipitation (ChIP) with antibodies against coilin and RNA Polymerase II (Pol II) in RPL knockdowns and discovered that coilin was no longer associated to gene loci and binding of Pol II along gene bodies was reduced. These results demonstrate that ribosomal proteins regulate the assembly and morphology of CBs. Finally, I combined proximity biotinylation with ChIP in a new method I have termed APEX-ChIP. The goal of APEX-ChIP is to understand how nuclear bodies interact with chromatin, by biotinylating molecular constituents surrounding the marker protein and thereby enhancing ChIP signals. As proof-of-principle, I show that APEX-ChIP is a viable method and use it on a well characterized nuclear body, the nucleolus. I performed nucleophosmin APEX-ChIP and show that I can detect DNA sequences corresponding to nucleolar organizing regions, while also revealing nucleophosmin at gene promoter regions. These discoveries represent the first unbiased and comprehensive CB components list that is functionally characterized for CB assembly. This is a crucial step in understanding the role that CBs and LLPS play in regulating the expression and 3D organization of genomes.

This document is currently not available here.