Characterizing isoform diversity in endothelial cells via proteogenomic methods that integrate long-read RNA sequencing and mass-spectrometry based proteomics

The process of alternative splicing enables one gene to produce multiple transcript isoforms that can produce proteins with distinct functions, expanding the diversity of the human proteome. The individual isoforms resulting from alternative splicing provide discrete levels of regulation that can modulate distinct cell phenotypes or cell fates. The evolution of RNA-sequencing technology has contributed to illuminating the prevalence of alternative splicing events, revealing that 95% of human genes are subjected to this process. However, the availability of comprehensive methods that delineate both transcript isoforms and associated protein isoforms remain limited. As a result, the isoform atlas remains incomplete for many tissue types, hindering knowledge of the degree at which alternative splicing impacts the proteome. In my dissertation work, I utilized innovative approaches to more effectively profile isoforms and their associated protein isoforms. My research benefitted from advancements in long-read RNA-sequencing technology, increasing resolution of the human transcriptome. I highlight in my first chapter, the development of an approach capitalizing on such RNA-sequencing advancements and integrating it with proteomics to create an isoform atlas. In my second chapter, I describe an application of this method to characterize the isoform landscape within endothelial cells, discovering novel protein isoforms for key markers of endothelial cell identity. I then review the roles of isoforms in directing cell fate decisions and their influence within gene regulatory networks. In my next chapter, I describe the knowledge of the potential roles of isoforms in development and how increases in resolution and throughput of long-read RNA-sequencing technologies were enabled by the introduction of a novel concatenation technique called MAS-Iso-Seq. These advances enabled the construction of an isoform atlas describing transcriptome dynamics during the process of early endothelial cell differentiation. Finally, I offer my perspectives on the state of the transcriptome analysis field and describe potential future directions for how such analysis can support precision medicine applications. Overall, my dissertation work supports enhanced profiling of isoform populations with specific focus on construction of an isoform atlas within endothelial cell types.

Chapter 4 refers to Mehlferber, M.M., Kuyumcu-Martinez, M., Miller, C.L. et al. Transcription Factors and Splice Factors—Interconnected Regulators of Stem Cell Differentiation. Curr Stem Cell Rep 9, 31–41 (2023). https://doi.org/10.1007/s40778-023-00227-2 with the associated license for reuse found in addition to this thesis.