Hochschulschrift:
Dissertation, Universität Freiburg, 2018
Anmerkungen:
Beschreibung:
Abstract: Alternative splicing (AS) is a fundamental regulatory process in gene expression, generating an immensely diversified transcriptome. Despite its importance, the spatiotemporal distribution of AS, its impact on development, and the machinery involved remain ill-defined, mainly due to the lack of tools to investigate AS in vivo. The aim of my PhD thesis was to investigate the role of splicing factors (SFs), the proteins that implement AS, during neuronal development and to generate tools to visualize endogenous AS events in vivo.<br><br>In the first part of this work, I performed an RNAi screen against neuronally expressed Drosophila SFs with human orthologs using the mushroom body (MB) as a model system. The screen identified nine SFs (16% of analyzed SFs) that elicited a clear MB phenotype, which I could confirm in mosaic genetic analysis using mutant alleles of the corresponding genes. The mutants displayed a variety of phenotypes suggesting pleiotropic functions for SFs in MB development. For example, depletion of half pint (hfp, the ortholog of the human Poly(U)-Binding Splicing Factor 60), a gene encoding a U2-related RNA-binding protein, resulted in fusion of the MB β lobes, whereas depletion of Splicing Factor 2 (SF2, the ortholog of the human Serine and Arginine-Rich Splicing Factor 1) led to segregation defects of the MB alpha/beta neurons. In addition, genetic characterization identified four mutants, including SF2, with non-cell-autonomous effects in mosaic genetic experiments, implying a role for SFs in the regulation of intercellular signaling pathways. In some cases, the phenotypes were also biased by sex (e.g. the hfp phenotype), an observation that is in line with the prominent role of sex-specific AS in several contexts, including neuronal morphogenesis and function. Furthermore, SFs identified to affect MB development were found to be dispensable in a different neuronal system, the horizontal cells, suggesting specific and context-dependent roles of AS in neuronal development. Speculations on molecular targets of the candidate SFs investigated in this work are based on literature research and include Dscam1 and shot.<br><br>In the second part of my thesis, I developed a method for monitoring AS events in vivo at physiological levels and for reporting their particular spatiotemporal patterns. The approach, which is adapted from the technique of dual-fluorescence splicing reporter minigenes, employs CRISPR/Cas9-aided genome editing and site-specific genomic integration to insert dual-tag splicing reporter constructs into the endogenous gene loci with minimal impact on the surrounding genome. Using this system, dual-tag splicing reporter cassettes have successfully been integrated for two genes of interest, dsx and Tango13, while analyses of the tissue distribution of splicing are currently in progress.<br><br>In summary, my work demonstrates an important role for SFs during MB development. Further detailed analysis of these factors and their targets will be important to identify mechanisms of action and biological functions during this developmental process. Furthermore, the possibility to visualize endogenous AS in vivo with my dual-tag splicing reporters should allow addressing important questions of transcript dynamics and AS regulation