DIORA family of proteins and functional methods of investigation

Abstract

This thesis includes projects in basic research that can be divided into two major subparts, one being development of a spatiotemporal genome engineering tool, BLU-VIPR, and the other being the functional investigation into the proteins from the gene family FAM167.To fully understand a biological system more information than the parts involved is needed, how they interact and organize into functional parts is essential for the functional output. Tools that can perturb the genome in a spatiotemporal fashion can help answer questions about how cells behave in space and time. The spatiotemporal tool BLU-VIPR utilizes the light-sensitive protein EL222 to induce production of gRNA upon blue light stimulation, thus allowing the formation of a functional gRNA-Cas complex. Using light to control gRNA production, instead of Cas expression, allows for a flexible system where multiple different CRISPR applications can be performed without cumbersome engineering. Since EL222 induces transcription from the C120 promoter by RNA polymerase II, the gRNA was flanked by self-cleaving ribozymes to allow for the precise excision of the gRNA from the transcript. Using ribozymes also allowed for the production of multiple gRNAs from one transcript as well as production of both gRNA and proteins. The functionality of BLU-VIPR was demonstrated in various CRISPR applications, including: CRISPRa, multiplexed CRISPRa, base editing, and CRISPR knockouts. BLU-VIPR enabled simultaneous induction of gRNA and protein, with very low leakiness observed in dark conditions. Additionally, the functionality of the system was demonstrated in vivo by CRISPR mediated knockouts in mouse T lymphocytes. This positions BLU-VIPR as a tool that can be used for investigating biological systems in a spatiotemporal fashion with high resolution, allowing for novel questions to be answered about the role of proteins in different contexts.Despite large efforts into sequencing the human genome, and by proteomics and transcriptomics proving that many genes are expressed, many proteins still have little or no functional description. One such poorly described gene family is FAM167, which contain two protein coding genes, FAM167A and FAM167B, with the proteins being denoted DIORA1 and DIORA2, respectively. To identify interactors for the DIORA proteins, we used BioID-based proximity proteomics and identified MRCK proteins as interactors to both DIORA1 and DIORA2. This was further validated in co-immunoprecipitation studies. For the DIORA1 protein, this interaction was further characterized by expressing mutants of the DIORA1 and MRCK proteins in co-immunoprecipitation experiments, resulting in three individual domains that allowed for interaction between the proteins. To determine the cellular function of DIORA proteins, we used CRISPRi to stably knockdown DIORA expression in neuroblastoma cell lines (DIORA1) and a melanoma cell line (DIORA2). Both DIORA1 and DIORA2 knockdown led to significant changes in transcriptional programs in the respective cell lines, with genes involved in epithelial to mesenchymal transition upregulated after DIORA1 knockdown and downregulated after DIORA2 knockdown. This was verified on a protein level in DIORA1 knockdown cells. Functional investigation of cellular motility was performed, and both DIORA1 and DIORA2 knockdown cells displayed altered invasion. DIORA2 knockdown cells also displayed altered expression of genes involved in oxidative phosphorylation, and had overall lower oxygen consumption, reduced mitochondrial RNA and DNA levels, and altered ADP/ATP ratio. These studies demonstrate an interest in further investigation of the role the DIORA proteins have in cellular motility, especially in the context of cancer cells with a potential mechanistic link between the MRCK kinases and DIORA proteins.List of scientific papersI. Light-induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo Diego Velasquez Pulgarin, Nathalie Pelo, Lin Ferrandiz, Tilen Tršelič, William A. Nyberg, Gary Bowlin, Alexander Espinosa Nucleic Acids Research, Volume 53, Issue 6, 11 April 2025, gkaf213. https://doi.org/10.1093/nar/gkaf213II. Autoimmunity-associated DIORA1 binds the MRCK family of serine/threonine kinases and controls cell motility Tilen Tršelič, Nathalie Pelo, Gregoire Martin de Fremont, Vaishnavi S. Iyer, Elina Richardsdotter Andersson, Vijole Ottosson, David Alexander Frei, Elisa Baas, William A. Nyberg, Guðný Ella Thorlacius, Lara Mentlein, Sanjaykumar V. Boddul, Ioana Sandu, Diego Velasquez Pulgarin, Ákos Végvári, Carmen Gerlach, Fredrik Wermeling, Maria Sunnerhagen, Björn Wallner, Alexander Espinosa, and Marie Wahren-Herlenius PNAS, October 3 2025, 122 (40) e2426917122. https://doi.org/10.1073/pnas.2426917122III. Identification of DIORA2 as a novel regulator of melanoma cell invasion and mitochondrial function Nathalie Pelo, Diego Velasquez Pulgarin, Lara Mentlein, William A. Nyberg, Vijole Ottosson, Ekaterina Zhuravleva, Gregoire Martin de Fremont, Tabassom Mohajershojai, Christina Gerstner, Liv Eidsmo, Elina Richardsdotter Andersson, Tilen Tršelič, Alexander Espinosa, Marie Wahren-Herlenius [Manuscript]</p