THE RNA EXOSOME: IMPLICATIONS FOR GENE EXPRESSION REGULATION AND NEUROLOGICAL PATHOLOGY Public
de Amorim, Julia (Fall 2023)
Abstract
The RNA exosome is a ten-subunit complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved and plays many roles in regulating gene expression and protecting the genome, including modulating the accumulation of R-loops at sites of transcription. The RNA exosome interacts with specific target RNAs for decay or processing via interacting proteins termed cofactors. Although the RNA exosome complex is routinely referred to as ubiquitously expressed, little is known about the tissue- or cell-specific expression of the RNA exosome complex or any individual subunit. Recently, missense mutations in genes encoding structural subunits of the RNA exosome have been linked to a variety of distinct neurological diseases, many of them childhood neuronopathies with at least some degree of cerebellar atrophy. Understanding how these missense mutations lead to the disparate clinical presentations that have been reported for this class of diseases necessitates investigation of how these specific changes alter cell-specific RNA exosome function. One possibility for how single amino acid changes could cause neurological disease is that the RNA exosome partners with cell- or tissue-specific protein cofactors. Here, we highlight recent studies that model pathogenic variants in RNA exosome subunits. In this study, we leverage publicly available RNA-sequencing data to analyze RNA exosome subunit transcript levels in healthy human tissues, focusing on those tissues that are impacted in exosomopathy patients described in clinical reports. We additionally employed a murine neuronal cell line (N2A) and performed immunoprecipitation of the RNA exosome subunit, EXOSC3, followed by mass spectrometry to obtain a snapshot of the RNA exosome interactome. We validated an interaction with DDX1, a putative RNA helicase. DDX1 plays roles in double-strand break repair, rRNA processing, and RNA metabolism. To explore shared functions of EXOSC3 and DDX1, we investigated the interaction after inducing DNA damage, and performed DNA/RNA immunoprecipitation followed by sequencing (DRIP-Seq) and RNA-seq on N2A cells depleted of either EXOSC3 or DDX1. These findings suggest that EXOSC3 and DDX1 function together in the absence of DNA damage to modulate spontaneous events such as RNA-DNA hybrid (R-loop) formation. Taken together, these analyses suggest that specific subunits could play impactful roles that affect the RNA exosome complex in gene expression and neurological pathology.
Table of Contents
CHAPTER 1: INTRODUCTION 1
THE RNA EXOSOME: A COMPLEX COMPLEX. 2
PATHOGENIC MISSENSE VARIANTS IN SUBUNIT GENES OF THE RNA EXOSOME ARE LINKED TO DISEASE. 3
EXOSC3 MUTATIONS IMPAIR RNA EXOSOME FUNCTION AND ORGANISM VIABILITY. 6
SHRF-CAUSING PATHOGENIC VARIANTS IN EXOSC2. 9
NOVEL EXOSC5 MUTATIONS IMPAIR RNA EXOSOME ACTIVITY. 10
CONCLUSIONS AND FUTURE PERSPECTIVES 11
FIGURE 1.1. PATHOGENIC MISSENSE VARIANTS IN STRUCTURAL SUBUNITS OF THE RNA EXOSOME CAUSE HUMAN DISEASE WITH DIVERSE CLINICAL PRESENTATIONS. 16
TABLE 1.1. EXOSOMOPATHY PATHOGENIC MISSENSE VARIANT MODELS 17
FIGURE 1.2. MODEL OF HOW PATHOGENIC MISSENSE MUTATIONS AFFECT THE RNA EXOSOME. 18
FIGURE 1.3. A PROPOSED MODEL OF HOW PATHOGENIC MISSENSE VARIANTS IN EXOSC GENES COULD CONTRIBUTE TO VARIATION IN CLINICAL PRESENTATION. 20
CHAPTER 2 21
ABSTRACT 22
INTRODUCTION 23
THE RNA EXOSOME COMPLEX PLAYS CRITICAL ROLES IN GENE EXPRESSION IN SUBCELLULAR COMPARTMENTS. 24
CLINICAL PHENOTYPES OF EXOSOMOPATHIES INCLUDE NEUROLOGICAL DEFECTS. 25
MATERIALS AND METHODS 28
GENOTYPE-TISSUE EXPRESSION (GTEX) PROJECT 28
RESULTS AND DISCUSSION 29
A COMPARATIVE ANALYSIS OF RNA EXOSOME SUBUNITS REVEALS DISPARATE REQUIREMENTS OF TRANSCRIPTS IN TISSUES. 29
FIGURE 2.1. THE RNA EXOSOME SUBUNIT TRANSCRIPTS ARE UBIQUITOUSLY EXPRESSED IN HUMAN TISSUE. 34
FIGURE 2.2. RNA EXOSOME SUBUNIT TRANSCRIPT LEVELS ARE HIGH IN THE CEREBELLAR HEMISPHERE/CEREBELLUM COMPARED WITH OTHER TISSUES. 36
CHAPTER 3 37
ABSTRACT 38
INTRODUCTION 39
RESULTS 42
PROTEOMICS REVEAL A SUITE OF EXOSC3 INTERACTORS. 42
PUTATIVE HELICASE DDX1 INTERACTS WITH THE RNA EXOSOME IN THE NUCLEUS. 43
THE INTERACTION BETWEEN EXOSC3 AND DDX1 DECREASES IN RESPONSE TO DNA DAMAGE. 45
DEPLETION OF EXOSC3 OR DDX1 RESULTS IN RRNA PROCESSING DEFECTS. 46
R-LOOPS ARE GLOBALLY REDUCED UPON DEPLETION OF EXOSC3 OR DDX1. 48
DISCUSSION 51
CHAPTER 3 FIGURES 59
FIGURE 3.1. RNA EXOSOME SUBUNITS CO-IMMUNOPRECIPITATE WITH TAGGED EXOSC3. 59
FIGURE 3.2: NOVEL EXOSC3/RNA EXOSOME INTERACTORS IDENTIFIED USING LIQUID CHROMATOGRAPHY COUPLED WITH TANDEM MASS SPECTROMETRY (LC-MS/MS). 61
FIGURE 3.3: DDX1 CO-IMMUNOPRECIPITATES WITH EXOSC3. 64
FIGURE 3.4: THE INTERACTION BETWEEN EXOSC3 AND DDX1 IS SENSITIVE TO DNA DAMAGE. 65
FIGURE 3.5: EXOSC3 AND DDX1 ARE ROBUSTLY DEPLETED BY SIRNA-MEDIATED KNOCKDOWN IN N2A CELLS. 67
FIGURE 3.6: DEPLETION OF EXOSC3 OR DDX1 RESULTS IN MISPROCESSING OF RRNA PRECURSORS. 70
FIGURE 3.7: DRIP-SEQ REVEALS THAT DEPLETION OF EXOSC3 OR DDX1 ALTERS R-LOOP REGIONS. 71
FIGURE 3.8: RNA-SEQ SHOWS THAT DEPLETION OF EXOSC3 OR DDX1 RESULTS IN MORE SHARED DECREASED TRANSCRIPTS THAN SHARED INCREASED TRANSCRIPTS. 73
FIGURE 3.9. FILTERING DRIP READS THROUGH RNA SEQUENCING REVEALED GENES THAT ARE SIMULTANEOUSLY AFFECTED BY DEPLETIONS OF EXOSC3 OR DDX1. 75
TABLE 3.S1. 76
FIGURE 3.S1. EXOSC3 CUSTOM-MADE ANTIBODY IS SPECIFIC, CELLULAR FRACTIONATION IS SUFFICIENT, AND THE INTERACTION BETWEEN EXOSC3 AND DDX1 IS IMPACTED BY LOSS OF RNA OR DNA. 77
FIGURE 3.S2. THE QUALITY OF THE PURIFIED RNA USED FOR THE NORTHERN BLOTS, RNA-SEQUENCING, AND DRIP-SEQUENCING ASSESSED BY 1% AGAROSE GEL AND HIGH SENSITIVITY SCREENTAPE ASSAY. 79
FIGURE 3.S3. IGV FOR R-LOOP REGIONS IN BAMBI IS ELEVATED IN CELLS DEPLETED OF EXOSC3 OR DDX1. 81
CONCLUSIONS AND FUTURE DIRECTIONS 83
EXOSOMOPATHIES AND THE BIOLOGICAL CONSEQUENCES OF PATHOGENIC MISSENSE MUTATIONS IN RNA EXOSOME SUBUNIT GENES. 85
NOVEL INTERACTIONS WITH THE RNA EXOSOME IN NEURONAL CELLS. 87
FIGURE 4.1. MODEL OF HOW THE RNA EXOSOME MAY IMPACT GENE EXPRESSION REGULATION AND NEUROLOGICAL PATHOLOGY. 94
REFERENCES 9
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