published_or_final_version / Medicine / Master / Doctor of Medicine

No description available.

No description available.

No description available.

No description available.

No description available.

Mammals receive more germline mutations from fathers than mothers. While the paternal bias in mutation has historically been attributed to errors in DNA replication during spermatogenesis, evidence suggests that in humans mutational mechanisms independent from cell division may play a more prominent role. Understanding how the ratio of paternal-to-maternal mutations, 𝛼, varies across animals differing in their gametogenic development, physiologies, and habitats can provide unique insights into the processes by which mutation arises in male and female germlines. To these ends, this thesis examines features of paternal mutation bias in dozens of amniote species using a combination of sequencing and evolutionary approaches. A direct way of measuring the strength of paternal mutation bias involves sequencing pedigrees of related individuals and detecting mutations arising in a single generation. In Chapter 2, we applied this approach to measure 𝛼 in olive baboons (Papio anubis) and humans. Strikingly, we estimated that in baboons 𝛼 = 4.5, similar to humans, despite baboons experiencing far fewer spermatogenic cell divisions than humans. A model of mutation based on cell division differences in the two species failed to explain this observation. Our results provide added evidence for non-replicative processes driving paternal bias in mutation and suggest that these causes are likely shared across mammals. In Chapter 3, we expanded our analysis to survey 𝛼 across 42 amniote species. We estimated 𝛼 from putatively neutral substitution rates of sex chromosomes and autosomes and found that in mammals, 𝛼 ranges up to 4 and correlates with generation times. In contrast, birds and snakes harbor a stable 𝛼 of roughly 2. These results are well predicted by modeling sex bias in mutation as a product of an early developmental phase when mutation occurs equally in both parents and a late phase after sexual differentiation when the male germline is more mutagenic. That the paternal mutation bias is widespread and occupies a narrow range of values suggests that it is caused by endogenous damage sources that are similar across species. Through a combination of pedigree sequencing and evolutionary techniques, this work demonstrates how a comparative approach across diverse taxa can shed light on the origins of sex-bias in germline mutation.

Long undecoded transcript isoforms (LUTIs) are a class of non-canonical mRNAs that repress gene expression by a combined mechanism of transcriptional and translational interference. Although this mechanism has been shown to be widespread in yeast, its prevalence in mammals has not been established. Using human embryonic stem cells (hESCs) differentiated into endoderm, mesoderm, and ectoderm lineages and further differentiation into polyhormonal cells, cardiomyocytes, and motor neurons, respectively, we obtained a comprehensive dataset through mRNA-seq, ribosome profiling, and quantitative mass spectrometry measurements. Our analysis revealed that LUTI-based regulation is context-dependent, with a total of 271 genes identified in ectoderm to motor neuron differentiation, 69 genes in mesoderm to cardiomyocyte differentiation, and 99 genes in endoderm to polyhormonal cell differentiation. Translational repression of LUTI candidates was found to be primarily dependent on upstream open reading frames (uORFs), while LUTI-based transcriptional repression displayed variability. This study enhances our understanding of gene expression and regulation during mammalian development and highlights the potential significance of LUTI-based regulation in the development of specific cell types or tissues. The findings lay the groundwork for further exploration into the role of LUTI- based regulation in other mammalian developmental programs and its potential implications for therapeutic targets in developmental disorders and diseases.

In an attempt to correlate genetic data with possible species delineation, this study investigates the presence of chromosomal variants between the North Atlantic bottle nose dolphin, Tursiops truncatus, and the North Pacific bottle nose dolphin, T. gilli. Blood samples were obtained from oceanaria in the United States. Location of capture was correlated with karyotype to compare chromosome morphology with geographic range.

Over 90% of human genes produce precursor mRNA (pre-mRNA) that undergoes splicing, an RNA processing mechanism. Alternative splicing (AS) of pre-mRNA allows a gene to generate multiple coding and non-coding isoforms by removing introns and ligating distinct exonic combinations. It is a mechanism that plays a major role in driving molecular diversity in mammals. This process is tightly regulated to determine the types and levels of protein products expressed in specific cellular contexts. Cis-acting splicing regulatory elements (SREs) found within the pre-mRNA are recognized and bound by RNA-binding proteins that either assist or interfere with the recruitment of the spliceosome. In the field of splicing, a long-standing goal has been to develop a “splicing code”, or a set of rules to understand the splicing patterns of a gene in a predictable manner. It is essential to highlight the significance of sequence context for SREs and the potential impact that distal intronic elements can have on splice site selection to better understand splicing. Given the importance of sequence context and the involvement of distal intronic regions in splicing, future approaches aimed at identifying SREs should consider these factors.This thesis will describe the process of the identification and validation of two novel distal intronic SREs located in critical disease exons. Importantly, these findings were made by combining experimental and computational approaches and through the development of a high-throughput SRE screening methodology. Chapter 1 will provide a general context for splicing, in particular AS, as an important mechanism among a plethora of RNA regulatory functions. The significance of AS regulation will be explored, as it plays a key role in the occurrence of physiological events and incorrect regulation can trigger disease. I will also introduce several methods used to study SREs, with experimental efforts primarily focusing on exonic and proximal intronic sequences. Additionally, as mis-splicing is associated with disease, there is a high interest in modulating splicing with novel therapeutic interventions, the development of which benefits from an increased understanding of SREs. Specifically, I will provide a landscape view of the splicing research field for the genes spinal muscular atrophy 2 (SMN2) and microtubule-binding protein tau (MAPT), given their relevance to our discoveries. As there is currently a paucity of high-throughput methods for studying SREs, especially those that allow for analysis of SREs in a near-native sequence context, I will introduce the CRISPR-Cas system (dCas13d) as a potential splicing modulator. This system will form the foundation for developing a tool to help us understand splicing regulation. Chapter 2 will discuss the discovery of a distal SRE regulating MAPT exon 10 splicing divergence in the primate lineage. Our lineage-specific AS analysis found that MAPT exon 10 shows a two-step evolutionary shift in the Catarrhine and hominoid lineages. The previously identified splicing regulatory elements cannot explain this evolutionary shift. Instead, a key splicing factor, muscleblind-like (MBNL), was found to be a major contributor to the observed splicing pattern divergence. Further mechanistic dissection revealed divergent, distal regulatory sequences in intron 10 that are recognized by MBNL. Based on this finding, we also demonstrated the potential of developing a therapeutically compatible strategy to target the MBNL binding sites by a steric hindrance to modulate exon 10 splicing effectively. Chapter 3 will discuss the development of a method allowing for a more unbiased, high-throughput screening of SREs, including those in the distal intronic regions. This method relies on the nuclease-inactive dRfxCas13d to modulate splicing. We use a dual-color fluorescent splicing reporter to identify the impact of splicing in a high-throughput manner. For our proof of concept, we use SMN2 exon 7 to identify SREs that influence the splicing of this exon and corroborate our findings with known SMN2 SREs. We performed a screen on the SMN2 dual-color splicing reporter using a gRNA library and obtained highly reproducible results. The screen also correctly identified gRNAs targeting known SREs, including the well-studied exonic regions and the downstream ISS-N1 element, the target of the ASO therapeutic known as nusinersen. Importantly, this screen also discovered novel splicing inhibiting gRNAs in a more distal region of the downstream intron, suggesting that a robust splicing enhancer was targeted. Previous studies likely overlooked this region due to its distance from the exon. This novel approach allows for the simultaneous screening of sizeable genetic regions using a large-scale gRNA library.