RNA can contain over 170 modified bases, adding another layer of information to transcripts and informing their functional outcomes in the cell. The function of many RNA modifications remains unknown, as well as how these modifications impact interactions between RNA and proteins. RNA binding proteins (RBPs) are a class of proteins that bind to RNA and are involved in all aspects of the RNA lifecycle. The ENCODE project has generated eCLIP data for 150 RBPs, revealing the binding locations of RBPs across the transcriptome. We leverage this dataset to compare where RBP binding overlaps with RNA modifications to infer interactions and identify functions of the modifications. We identified novel RBP interactions with the common mRNA modifications pseudouridine and m6A. m6A has been found to be dysregulated in cancer and is being investigated as a target for new therapeutics. To interrogate patterns of m6A deposition in cancer, m6A-eCLIP was performed on the NCI-60 cell lines. We identified common m6A peaks in oncogenes that could be targets for new treatments. This work will allow further studies of RNA modification-RBP interactions and their role in cancer progression.

CRISPR-Cas13 has been developed as a tool for genome editing by enabling precise RNA manipulation without permanently altering the genome. While its potential in therapeutics and diagnostics is promising, Cas13d’s off-target activities have limited its further use. To enhance our understanding of Cas13d’s binding specificity, we performed a massively parallel filter-binding assay probing the impact of mismatches in the target RNA on binding. Our results revealed that Cas13d binding is greatly affected by the specific position of the mutations in the target sequence, with less dependence on the type of mutation. Mismatches in the 5’ region of the target sequence were well tolerated, while those in the central region abrogated binding. These results emphasize the requirement for a complementary sequence in the central region for specific Cas13d binding. Additionally, this study expands the filter-binding method as a valuable tool for analyzing the binding specificity and off-target effects of Cas13d. Thus, our work lays a solid foundation for future high-throughput profiling of Cas13d binding and applying Cas13d for safe and effective transcriptional perturbation.

Stress granules (SG) form during cellular stress and have been linked to neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Strategies to modulate SGs have the potential to yield insights into their role in pathophysiology. We performed a high-content screen to identify small molecules that alter SG properties in proliferative cells and iPSC-derived motor neurons (iPS-MNs). One major class of hit compounds are nucleic acid intercalating molecules that disrupt recruitment of ALS-associated RNA-binding proteins (RBPs), such as TDP-43 and FUS/TLS, into SGs and reduce accumulation of cytoplasmic TDP-43 in iPS-MNs from ALS patients. These findings corroborate our biochemical results indicating that SGs accumulate ALS-associated RBPs in their shells in an RNA-dependent manner. Thus, SGs contribute to persistent accumulation of TDP-43, and nucleic acid intercalating compounds represent a promising new therapeutic approach for treating ALS/FTD.

My thesis work is aimed at integrating high-throughput biochemical readouts to understand the effect that particular RNA-binding proteins have on their targets' metabolism. I studied several RNA-binding proteins in diverse model organisms. Along the way I have gained insight into the characteristics of target sites for two families of proteins: Argonaute and RBFOX proteins and learned about novel mechanisms they use to control their targets' fate.

In this dissertation, I will present three of my articles that summarize the methods and content of my work. In the first chapter I give an overview of methods I used to quantify the ideal depth of coverage for sequencing experiments in order to sufficiently capture a desired level of complexity. This included the development of novel computational approaches to quantify RNA abundance and splicing with sequencing tools. This was applied to a human model of prostate cancer, LNCaP cells upon stimulation with an androgen compound.

To closely examine mechanisms of miRNA regulation, we generated genome wide maps of the Argonaute protein ALG-1's binding in C. elegans. We found that there exists a large potential for non-canonical associations of ALG-1/miRNA complexes with their targets. Among the surprises we encountered, ALG-1 binds in coding exons but does not seem to repress gene expression when bound there, and we also found an auto-regulatory repression by ALG-1 on miRNA pathway components.

Finally, I turned my attention to the RBFOX family of proteins that is known for their role in RNA splicing regulation. In the work presented in chapter 4, we elucidated a new molecular mechanism whereby RBFOX proteins can regulate RNA splicing from very distant sites. These RNA-bridges, as we have called them because they link RNA regulators with regulated sites via RNA structures, appear to be a common feature of alternatively spliced exons and the regulation of RNA structures like this may be important for dictating splicing outcomes. The application of this knowledge that distant binding sites are functional and that they are mediated by RNA structures is immediately relevant to the design of novel therapeutics for diseases that arise from defects in RNA splicing.

To put the goals of my thesis broadly, I approach two questions: what are the mechanisms of RNA-binding protein targeting, and what are the effects of RNA-binding proteins on their direct targets?

Cellular behavior is dictated by numerous factors including cell-type and environmental-dependent gene expression as well as somatically acquired mutations in cancer. The goals of my thesis research are to employ high throughput techniques and informatics to assay gene function in the regulation of disease-relevant phenotypes. First, we developed a screening platform to combine image-based readouts with the easy-to-use pooled CRISPR knockout approach. Pooled CRISPR methods allow for the genetic knock-out of tens of thousands of genes in a single pool of cells, but are limited thus far to bulk-selected phenotypes including proliferation rates and FACS-based screening. In this work, we adapted a microraft array platform and developed automated imaging and analysis programs to screen over 1,000 RNA binding proteins (RBPs) for their role in regulating the abundance of cytoplasmic stress granules, a cellular phenotype with implications in neurodegeneration. Second, to understand the role of somatic mutations in splicing factor (SF) proteins to drive cancer phenotypes, we developed a human isogenic induced pluripotent stem cell (iPSC) model of the canonical \emph{\U2AF1 S34F} and \emph{SRSF2 P95L} mutations. We found that iPSC-derived hematopoietic stem/progenitor cells (iPSC-HSPCs) have a reduced ability to terminally differentiate into mature blood cells, thus reproducing the cancer phenotype found in patients with Myelodysplastic Syndromes (MDS). An integrative RNA-Seq and eCLIP analysis these cells revealed an that alternative splicing of \emph{GNAS} is a direct target of mutant SFs. Functional studies confirmed that expression of the cancer-associated isoform, \emph{GNAS-L} reproduced our disease phenotype in normal cells, and correction of SF-mutant cells to \emph{GNAS-S} rescued the disease phenotype back to normal levels. \emph{GNAS} is a major regulator of cell signaling, and also contains point mutations in a subset of MDS patients. Mutational analysis of patient data and biochemical work on purified forms of this protein suggest usage of \emph{GNAS-L} in addition to the \emph{GNAS R201H} point mutation cooperate to induce overactivation of G-protein signaling in cancer cells. This work has identified a novel cellular function of \emph{GNAS} isoform regulation, and opened up the door for G protein signaling as a potential therapeutic avenue for SF-mutant MDS.

Alternative splicing (AS) generates isoform diversity critical for cellular identity and homeostasis in multicellular life. Although AS variation has been observed among single cells for a few events, little is known about the biological significance of such variation. We developed Expedition, a computational framework consisting of outrigger, a de novo splice graph transversal algorithm to detect AS; anchor, a Bayesian approach to assign modalities and bonvoyage, a visualization tool using non-negative matrix factorization to display modality changes. Applying Expedition to single iPSCs undergoing neuronal differentiation, we discover up to 20% of AS exons exhibit bimodality and are flanked by more conserved introns harboring distinct cis-regulatory motifs. Bimodal exons constitute the majority of cell-type specific splicing, are highly dynamic during cellular transitions, preserve translatability and reveal intricacy of cell states invisible to global gene expression analysis. Systematic AS characterization in single cells redefines our understanding of AS complexity in cell biology.

While the cells that comprise an organism contain largely the same DNA content, the RNA content of cells varies widely among different cell types and healthy vs. diseased cells. The portions of the genome that are transcriptionally active define cell identity and a highly-regulated set of RNA processing steps mediate the generation of the cellular RNA pool. A host of conditions are linked to dysfunctional RNA processing, and the development of means to measure and manipulate cellular RNA has been a persistent concern in the biotechnological fields. This thesis focuses on the development flexible and programmable means to measure and manipulate RNA in living cells using both synthetic and repurposed natural systems.

RNA-binding proteins (RBPs) are critical regulators of post-transcriptional gene expression and aberrant RBP-RNA interactions can cause cancer. However, RBPs are often overlooked as therapeutically relevant targets because unlike transcription factors, altered RBP activity is more frequently caused by changes in the expression levels of RBPs and their underlying targets, as opposed to somatic mutations. It has therefore been challenging to systematically evaluate the function of RBPs in disease. We addressed the lack of characterization of RBPs in cancer by developing an approach to interrogate the function of RBPs using pooled CRISPR-Cas9 screening. We identified 57 putative RBPs with distinct roles in supporting MYC-driven oncogenic pathways and we found that disrupting YTHDF2-dependent degradation of its target transcripts triggers apoptosis of MYC-dependent cancer cells and tumors. Next, we profiled YTHDF2 function using enhanced CLIP (eCLIP) and m6A-seq analysis, which revealed extensive interactions with mRNAs encoding MAPK pathway genes. We found that mRNA stabilization of upstream MAPK pathway genes drives epithelial-to-mesenchymal transition in MYC-dependent cells and is accompanied by profound oxidative cellular stress due to increased protein synthesis. We next explored the cellular stress response elicited by YTHDF2 depletion by surveying the activation of the intrinsic and extrinsic apoptotic pathways. Loss of YTHDF2 caused activation of the unfolded protein response and subsequent mitochondrial dysfunction in MYC-dependent cells, which are especially sensitive to increases in oxidative stress. Finally, we determined that PRSS23 mRNA stabilization is necessary for driving apoptosis by promoting TCF12-mediated transcription of cancer lineage-specific transcripts and translation factors. Thus, this dissertation highlights the therapeutic relevance of RBPs by uncovering the critical role of YTHDF2 in counteracting the global increase of mRNA synthesis in MYC-driven cancers.

Emerging genomic technologies that measure spatial information about RNA molecules promise to shed light on cell biology and function. However, most analytical techniques have primarily concentrated on spatial relationships at the multicellular and cellular scale without fully tapping into single-molecule spatial information. To address this gap, I introduce Bento, a toolkit designed for discerning spatial relationships at the subcellular scale. Bento incorporates a suite of statistical and machine learning methods within an intuitive Python programming interface, emphasizing the FAIR data management principles. To showcase its capabilities, I utilized Bento to study RNA localization changes in doxorubicin-treated cardiomyocytes profiled with spatial transcriptomics. Our findings reveal that doxorubicin-induced stress leads to the depletion of disease-associated genes in the endoplasmic reticulum, along with expression changes previously associated with doxorubicin-induced cardiotoxicity. This places the endoplasmic reticulum as a pivotal subcellular structure in the response to doxorubicin treatment. In essence, Bento emerges as a potent toolkit for the subcellular analysis of spatial transcriptomics data, paving the way for the discovery of new spatial relationships between subcellular structures and molecules. Furthermore, I have created a framework tailored to streamline image processing for spatial transcriptomics data called spotfish. Similar to Bento's ethos, spotfish is built in alignment with FAIR principles and leverages open-source standards like the Nextflow workflow language and Open Microscopy Environment file formats. Collectively, Bento and spotfish empower researchers to harness spatial transcriptomics technologies, enabling more comprehensive exploration of the spatial and molecular organization of cells at an unprecedented throughput.

The process of protein targeting to mitochondria is complex and relies on both polypeptide and mRNA mediated pathways in a partially redundant manner. Mitochondria associated RNA-binding proteins (RBPs) have emerged as key facilitators of post-transcriptional regulation of nuclear-encoded mitochondrial mRNAs (NEMmRNAs), often by enhancing protein targeting to mitochondria. With only a few examples described in humans, I set out to identify RBPs with a role in post-transcriptional regulation of NEMmRNAs. Through a systematic analysis of the ENCODE collection of eCLIP datasets mapping the binding sites of over a 150 RBPs, I identified several RBPs with RNA-target sets enriched in NEMmRNAs, of these I selected LARP4 for further analysis. From data produced in multiple human cell lines, I show that the RNA-target set of LARP4 is particularly enriched in mRNAs that encode oxidative phosphorylation proteins (OXPPs) and mitochondrial ribosome proteins (MRPs). I generate LARP4 knockout cell lines to study the consequences of LARP4 depletion. By immunoblot analysis and quantitative proteomics analysis, I show that OXPPs and MRPs are present in reduced abundance in LARP4 knockout cells. Interestingly, cell fractionation experiments showed that the depletion of OXPPS and MRPs was more pronounced in mitochondrial fractions than in whole cell fractions, suggesting defect in protein targeting to mitochondria. Additionally, I show that the depletion of these proteins essential to oxidative phosphorylation has a functional impact on oxidative phosphorylation rates as well as energy dependent cellular processes such as cell proliferation and translation. Furthermore, I show these phenotypes are ameliorated by LARP4 re-expression. Together these data support a novel functional role of LARP4 in post-transcriptionally regulating NEMmRNAs to promote their expression and support oxidative phosphorylation function.