Normal mRNA turnover is essential for genetic regulation within cells. The E. coli RNA degradosome, a large multi-component protein complex which originates through specific protein interactions, has been referred to as the “RNA decay machine” and is responsible for mRNA turnover. The degradosome functions to process RNA and its key components have been identified. The scaffold protein is RNase E and it tethers the degradosome to the cytoplasmic membrane. Polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlB helicase) and the glycolytic enzyme enolase associate with RNase E to form the degradosome. Polyphosphate kinase associates with the degradosome in substoichiometric amounts, as do the molecular chaperones DnaK and GroEL. The role of DnaK as well as that of enolase in the RNA degradosome is unknown. Very limited research has been conducted on the components of the RNA degradosome under conditions of stress. The aim of this study was to understand the role played by enolase in the assembly of the degradosome under conditions of stress, as well as investigating the protein levels of molecular chaperones under these conditions. The RNA degradosome was successfully purified through its scaffold protein using nickel-affinity chromatography. In vivo studies were performed to investigate the protein levels of DnaK and GroEL present in the degradosome under conditions of heat stress, and whether GroEL could functionally replace DnaK in the degradosome. To investigate the recruitment of enolase to the degradosome under heat stress, a subcellular fractionation was performed to determine the localization of enolase upon heat shock in vivo. The elevated temperature resulted in an increased concentration of enolase in the membrane fraction. To determine whether there is an interaction between enolase and DnaK, enolase activity assays were conducted in vitro. The effect of DnaK on enolase activity was measured upon quantifying DnaK and adding it to the enolase assays. For the first time it was observed that the activity of enolase increased with the addition of substoichiometric amounts of DnaK. This indicates that DnaK may be interacting with the RNA degradosome via enolase.

Genetic discoveries have highlighted the role of gene expression dysregulation in both rare and common diseases. In particular, a large number of chromatin modifiers, transcription factors, and RNA-binding proteins have been implicated in neurodevelopmental diseases, including epilepsy, autism spectrum disorder, schizophrenia, and intellectual disability. Elucidating the disease mechanisms for these genes is challenging, as the encoded proteins often regulate thousands of downstream targets. In Chapter 2 of this thesis, we describe the use of single-cell RNA-sequencing (scRNA-seq) to characterize a mouse model of HNRNPU-mediated epileptic encephalopathy. This gene encodes a ubiquitously expressed RNA-binding protein, yet we demonstrate that reduction in its expression leads to cell type-specific transcriptomic defects. Specifically, excitatory neurons in a region of the hippocampus called the subiculum carried the strongest burden of differential gene expression. In Chapter 3, we use scRNA-seq to identify convergent molecular and transcriptomic features in four different organoid models of a cortical malformation called periventricular nodular heterotopia. In Chapter 4, we build on these successes to propose a high-throughput drug screening program for neurodevelopmental genes that encode regulators of gene expression. This approach—termed transcriptomic reversal—attempts to identify compounds that reverse disease-causing gene expression changes back to a normal state. Finally, in Chapter 5, we focus on the role of synonymous codon usage in human disease. Codon usage can affect mRNA stability, yet its role in human physiology has been historically overlooked. We use population genetics approaches to demonstrate that natural selection shapes codon content in the human genome, and we find that dosage sensitive genes are intolerant to reductions in codon optimality. We propose that synonymous mutations could modify the penetrance of Mendelian diseases through altering the expression of disease-causing mutations. In summary, the work in this thesis broadly focuses on the role of gene expression dysregulation in disease. We provide novel frameworks for interrogating disease gene expression signatures, prioritizing mutations that may alter expression, and identifying targeted therapeutics.

Amyloids are fibrous protein aggregates associated with age-related diseases, such as Alzheimer’s disease and Parkinson’s disease. The role of amyloids in the etiology of neurodegeneration is debatable, but genetic and molecular evidence supports a causative relationship between amyloidogenesis and disease. Amyloidogenic proteins are constitutively expressed throughout the lifespan of an organism, and yet only become pathogenic in certain situations. This led to a hunt to understand how amyloidogenic proteins could be modified in order to become aggregation-prone. One possibility that has garnered attention is phosphorylation, primarily because several amyloid aggregates such as tau and α-synuclein are often highly phosphorylated in disease. However, the contribution of phosphorylation to disease progression remains unclear.While amyloid aggregates are typically described as irreversible and pathogenic, some cells utilize reversible amyloid-like structures that serve important functions. One example is the RNA-binding protein Rim4 which forms amyloid-like assemblies that are essential for translational control during S. cerevisiae meiosis. If Rim4 is unable to translationally repress its mRNA targets, cells mis-segregate chromosomes during meiosis resulting in aneuploid gametes. Importantly, Rim4 amyloid-like assemblies are disassembled in a phosphorylation-dependent manner at meiosis II onset which allows previously repressed transcripts to become translated. In Chapter 1, I describe the significance and complexity of protein phosphorylation as it relates to disease-associated amyloids and why Rim4 is an ideal model for studying this phenomenon. The objective of this thesis is to examine the mechanisms underlying clearance of Rim4 amyloid-like assemblies. The work described in Chapter 2 focuses on identifying co-factors that mediate clearance of amyloid-like assemblies in a physiological setting. I demonstrate that yeast 14-3-3 proteins, Bmh1 and Bmh2, bind to Rim4 assemblies and facilitate their subsequent phosphorylation and timely clearance. Furthermore, distinct 14-3-3 proteins play non-redundant roles in facilitating phosphorylation and clearance of amyloid-like Rim4. In Chapter 3, I explore the mechanism underlying 14-3-3 contribution to Rim4 amyloid-like disassembly. I find that 14-3-3 proteins are critical for the interaction between Rim4 and its primary kinase Ime2, thus facilitating downstream multi-site phosphorylation of Rim4. In Chapter 4, I explore additional roles for 14-3-3 proteins in general protein aggregate homeostasis. I find that 14-3-3 mutants exhibit greater protein aggregate burdens. Additionally, 14-3-3 mutants accumulate ubiquitinated proteins and are sensitized to proteasome mutations, suggesting a role for 14-3-3 proteins in proteasome function. Collectively, the studies described in this thesis support a protective role for 14-3-3 proteins in protein aggregation that may have implications for amyloid biology in human disease.

Fused in Sarcoma (FUS) is a nuclear RNA binding protein functioning in a number of essential cellular processes such as RNA processing and DNA damage response. Mutations in FUS gene contribute to 5% of familial Amyotrophic Lateral Sclerosis (ALS) characterized by FUS protein cytoplasmic aggregation. Despite efforts have been made in the past decade, mechanisms of FUS aggregates to induce cytotoxicity are not fully understood. In addition, wild-type FUS protein has been found mis-localized to cytoplasm in sporadic ALS and Frontotemporal Dementia (FTD) patients with unclear mechanisms. Here, we aimed to address the functional consequences of ALS mutant FUS aggregation and investigate the mechanisms of wild-type FUS cytoplasmic translocation. This dissertation is divided into three parts: In the first part, we review pathophysiological mechanisms of FUS and other ALS mutant genes which induce cell death via disrupting six major cellular processes: mRNA processing, non-sense mediated decay, mitochondrial functions, nucleocytoplasmic transport, autophagy and DNA damage response. In the second part, we aimed to understand the functional consequences of RNA sequestration by FUS aggregates. We performed RNA immunoprecipitation against exogenous or endogenous FUS in the transfected cell lines and mutant FUS ALS patient fibroblasts to isolate RNAs associated with wild-type or ALS mutant FUS. Next, we analyzed the isolated RNAs using poly(A+) RNA-specific sequencing 3’READS and RT-qPCR, and we found many nuclear-encoded respiratory chain complex mRNAs are top-enriched transcripts associated with ALS mutant or overexpressed wild-type FUS. We further demonstrated that respiratory chain complex mRNAs are sequestered in mutant FUS cytoplasmic aggregates and the encoded protein expression levels are suppressed. Finally, we showed that knockdown of respiratory chain complex proteins encoded by FUS-sequestered transcripts can recapitulate mitochondrial dysfunction observed in FUS-transfected cell lines. Our findings in the second part thus provides a novel mechanism by which ALS mutant FUS, as well as overexpressed wild-type FUS, to induce mitochondrial dysfunction via preferential sequestration of respiratory chain complex mRNAs. The third part focuses on understanding pathways affecting FUS nucleocytoplasmic distribution. By using pharmacological treatments and immunofluorescence, we found that nuclear RNA transcription, export and decay substantially modulate nucleocytoplasmic distribution of wild-type FUS protein. Moreover, we report that FUS antibodies used in immunofluorescence significantly affect the results of nucleocytoplasmic ratio quantification. Intriguingly, we observed altered serine-2/-5 phosphorylation on RNAPII CTD as well as reduced number of nascent transcripts in sporadic ALS patient cells, indicating aberrant transcriptional activity related to cytoplasmic accumulation of nuclear RNA binding proteins. Our findings in the third part provide insights to the importance of nuclear RNA metabolism in modulating FUS localization. We also addressed the inconsistent results reported in previous studies regarding FUS nucleocytoplasmic distribution in response to stress. Altogether, these findings suggest proof-of-principle mechanisms of FUS toxic function and aberrant localization linked to ALS and FTD disease spectrum.

The study of protein synthesis, and the study of gene expression in general, has accelerated in recent years. Following the advent of next-generation RNA sequencing, powerful library preparation paradigms were developed to capture regulatory activity on a genome-wide scale. In particular, ribosome profiling has emerged as a widely-used measurement of translation. In this method, the state of ribosome association across the transcriptome is obtained by isolation and sequencing of the regions of RNA bound by ribosomes, revealing a snapshot of ribosome positions from which gene-specific densities can be calculated. In combination with RNA sequencing for a measurement of baseline transcription in the same samples, ribosome profiling offers a metric of “translation efficiency”, or TE, corresponding to the average ribosome load per given transcript. Ribosome profiling has advanced the study of translation considerably. However, low throughput in the generation of ribosome profiling and RNA sequencing libraries limits the scale of the experiments that can be performed, while issues in the interpretation of aligned ribosome-protected footprints complicate their analysis, especially in systems of complex regulation. The analysis of such regulatory systems would be greatly aided by a high-throughput sequencing method that can capture translational regulation, but current methods of measuring genome-wide translation are inherently limited in scale. This thesis addresses the key issues presented above in separate chapters. Chapter 2 discusses the analysis of elongation and initiation from ribosome profiling and RNA sequencing data in a mouse model of Fragile X Syndrome. In this chapter, several methods of measuring and modeling variability in the distribution of ribosomes along a coding sequence are used alongside analyses of differential RPF and RNA abundances and their ratio, RFApm, which we distinguish from TE to emphasize its dependence on factors other than initiation rate. The chapter summarizes current information regarding the observed effects of FMRP, and proposes a model congruent with these observations and more-recently published studies. Chapters 3 and 4 present approaches to modeling or inferring translational regulatory networks, either by a novel library preparation paradigm or computational inference from publicly-available data. Chapter 3 presents riboPLATE-seq, a high-throughput RNA-seq library construction method based on the existing PLATE-seq method. The method recapitulates significant findings from ribosome profiling and RNA sequencing at a fraction of the per-sample cost, with further advantages in scalability, and could be implemented in a large-scale screen of translational regulators to create a network of their specific targets. Chapter 4 presents an approach to inferring translational regulation from integrative analysis of public ribosome profiling and RNA sequencing data, tailoring the powerful inference engine ARACNe to measure translational interactions. This yields a comprehensive network of translational regulation, assigning target genes to the set of RNA-binding proteins.

Indiana University-Purdue University Indianapolis (IUPUI) / In an effort to advance the methodology in analyzing RNAPII protein-protein interaction network and to determine the role of the CTD in controlling RNAPII transcription, we devised a method to specifically isolate the CTD-associated and CTD-less RNAPII to identify the proteins that interact with both the CTD and the globular core of RNAPII using novel purification scheme coupled to quantitative proteomics.

Cell type-specific alternative splicing (AS) of pre-mRNA regulated by RNA-binding proteins (RBPs) is widespread, but particularly prominent in the brain, driving gene isoform differences between a diverse range of neuron types. While several AS programs have been shown to be critical to the function of particular neuron types, previous studies have usually been limited to one or a few RBPs and cell types, resulting in a piecemeal understanding of these regulatory patterns. Towards a comprehensive view of the neuron type-specific AS regulatory landscape, we apply current computational and experimental methods to survey neuronal AS, infer its regulation by hundreds of RBPs, and experimentally validate regulatory predictions. In Chapter 1, we examine AS in 133 transcriptomic cell types of mouse cortical neurons defined by single-cell RNA sequencing (scRNA-seq) and define neuron type-specific exons and some of their likely regulators. In Chapter 2, we leverage the rich transcriptomic dynamics of the cortical neuron dataset to systematically infer splicing regulatory network and predict RBP activity on the cell type level. We use the information theory-based method ARACNe to reverse engineer RBP-target regulatory networks and VIPER to infer differential RBP activity across neuron types in a workflow we call Master Regulator analysis of Alternative Splicing (MR-AS). RBP regulons predicted by MR-AS are consistent with high-confidence lists of RBP targets and are supported by motif and CLIP read distribution analyses. Estimation of cell type-specific RBP activity using the predicted regulons shows the expected decreases in RBP KO samples. Chapter 3 focuses on two neuron type-specific AS regulatory programs as case studies, which we validate in vitro using embryonic stem cell (ESC)-derived neuron types. Elavl2 was predicted to drive neurons towards an MGE interneuron-specific AS profile. Elavl2 knockout in ESC-derived MGE interneurons causes modulation of exon inclusion consistent with the predicted regulation of MGE interneuron AS, shifting their splicing profiles towards those of CGE interneurons. We also identified a module of exons that show consistent AS between long- and short-projection neurons across multiple neuronal classes, which are shifted in the expected direction when ESC-derived interneurons are transcriptionally reprogrammed to reflect a long-axon globus pallidus-like neuronal identity. In Chapter 4, we use the RBP regulons to predict RBP activity on a single-cell level and examine its variability, leading us to identify both neuron type-specific AS programs and a neuron type-orthogonal gradient of activity (NTOG). Exons associated with responses to neuronal depolarization and long-term potentiation show a gradient of inclusion across the NTOG, suggesting it may reflect differential activation of activity-dependent AS programs of the assayed neurons. Together, the results described in this thesis demonstrate the validity and broad utility of the inferred AS regulatory networks as a resource for elucidating RBP splicing regulation differences and their functional impact across neuron types.

Budding yeast cells have the capacity to adopt distinct physiological states depending on environmental conditions. Vegetative cells proliferate rapidly by budding while spores can survive prolonged periods of nutrient deprivation and/or desiccation. Whether or not a yeast cell will enter meiosis and sporulate represents a critical decision which could be lethal if made in error. Most cell fate decisions, including those of yeast, are understood as being triggered by the activation of master transcription factors. However, mechanisms that enforce cell fates post-transcriptionally have been more difficult to attain. Here, we perform a forward genetic screen to determine RNA-binding proteins that affect meiotic entry at the post-transcriptional level. Our screen revealed several candidates with meiotic entry phenotypes, the most significant being RIE1 which encodes an RRM-containing protein. We demonstrate that Rie1 binds RNA, is associated with the translational machinery, and acts post-transcriptionally to enhance protein levels of the master transcription factor Ime1 in sporulation conditions. We also identified a physical binding partner of Rie1, Sgn1, which is another RRM (RNA Recognition Motif)-containing protein that plays a role in timely Ime1 expression. We demonstrate that these proteins act independently of cell size regulation pathways to promote meiotic entry. We propose a model explaining how constitutively expressed RNA-binding proteins, such as Rie1 and Sgn1, can act in cell-fate decisions both as switch-like enforcers and as repressors of spurious cell fate activation. Chapter 1 serves as a brief overview of the importance cell fate decisions and details how sporulation in the budding yeast Saccharomyces cerevisiae can be used as a model to understand the pathways and mechanisms underlying these decisions. This chapter focuses on the importance of the meiotic master regulator IME1 and the different effectors of regulation that govern its expression at the transcriptional and post-transcriptional levels. Chapter 2 describes the significance of RNA binding proteins and how they can influence cell fate decisions with a focus on cell cycle modifications that shift mitosis to meiosis. Chapter 3 explains the methodology that I used to discover two RNA-binding proteins that play key roles in meiotic entry: Rie1 and Sgn1. Chapter 4 describes my work to dissect the pathways governed by Rie1 and Sgn1. Chapter 5 discusses the potential mechanisms by which Rie1 and Sgn1 could drive entry into meiosis. Collectively, the studies described in this thesis demonstrate that Rie1 and Sgn1 affect the cell fate decision to enter meiosis in budding yeast by activating as translational activators of IME1.

Bacteria live under constant assault by bacteriophages and have evolved a diverse array of defense strategies. CRISPR-Cas systems are prokaryotic adaptive immune systems that rely on RNA-guided binding for the recognition and degradation of invading nucleic acids. Intriguingly, some bacteria also encode divergent CRISPR-Cas systems that can bind to — but cannot degrade — target nucleic acids. In this dissertation, I describe the study of nuclease-deficient CRISPR-Cas systems alongside the evolutionary pressures that led to their persistence in bacterial genomes. I present experimental data for the existence of CRISPR-associated transposons (CASTs) that utilize the RNA-guided DNA binding ability of Type I-F CRISPR-Cas systems to direct transposition to new target sites in a heterologous Escherichia coli host. This RNA-guided DNA integration pathway can tolerate large cargos of up to 10 kilo-base pairs in size, and is highly specific for the programmed target site, as determined by deep sequencing experiments. We further reveal the physical link between the CRISPR-Cas and transposition machineries through biochemical experiments and by determining cryo-EM structures of the transposition protein TniQ in complex with the CRISPR-Cas effector. After bioinformatic analyses and experimental validation we established an array of twenty diverse CAST systems for which a subset works completely orthogonally. This dataset revealed the modular nature of CASTs by showcasing the horizontal acquisition of targeting modules and by characterizing a system that encodes both a programmable, RNA-dependent pathway, and a fixed, RNA-independent pathway. Further analysis of the transposon-encoded cargo genes uncovered the striking presence of anti-phage defense systems, suggesting a role in transmitting innate immunity between bacteria. Finally, we exploit high-throughput screening assays to determine the specific sequence and spacing requirements of the transposon ends, and use this knowledge to develop a CAST-mediated endogenous gene-tagging approach. Intriguingly, our experiments uncover the involvement of a previously unknown cellular protein, integration host factor (IHF), which is critical for transposition of VchCAST, but not other homologous systems. Collectively, the work presented in this dissertation describes the discovery of RNA-guided DNA integration employed by CASTs, substantially advances our biological understanding of these systems, and expands the suite of RNA-guided transposases for programmable, large-scale genome engineering.

T-cell Intracellular Antigen-1 (TIA1) is a multi-domain RNA-binding protein involved in stress granule formation and implicated in neurodegenerative diseases. TIA1 contains three RNA recognition motifs (RRMs), which are capable of binding nucleic acids, and a C-terminal intrinsically disordered prion-related domain (PRD), which plays a role in promoting liquid-liquid phase separation. Motivated by our previous findings indicating that RRMs 2 and 3 exhibit a well-ordered structure in the oligomeric full-length form, whereas RRM1 and PRD demonstrate a propensity for phase separation, the present work in this dissertation aims to investigate the functional competence of the oligomeric state and its binding capabilities. Moreover, the study explores the effects of ligand binding on oligomerization dynamics and potential alterations in protein conformation primarily using solid-state NMR methods. The NMR data show that ssDNA binds to full-length oligomeric TIA1 primarily at RRM2, but also weakly at RRM3, and Zn2+ binds primarily to RRM3. The binding of Zn2+ and DNA was reversible and without the formation of amyloid fibrils. The addition of Zn2+ caused the TIA1:DNA complexes to collapse, indicating that Zn2+ may play a regulatory role by shifting the nucleic acid binding off RRM3 and onto RRM2 by occupying various “half” binding sites on RRM3 and introducing a mesh of crosslinks in the supramolecular complex. Furthermore, this dissertation presents an investigation into the interdomain interactions between RRM2 and RRM3, facilitated by the successful preparation of segmentally labeled protein samples using the trans-splicing approach. The results confirm the hypothesis that Zn2+ can bring RRM2 and RRM3 closer together by crosslinking different monomers, as evidenced by the observation of enhanced NMR signals from heteronuclear correlations around the Zn2+ binding sites. In conclusion, studying the structure of full-length TIA1 oligomers is expected to reveal the mechanisms by which an RNA regulatory protein assembles and binds to its biologically relevant ligands while preserving a highly ordered oligomeric structure.