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The BEACH domain containing proteins (BDCPs) represent a highly conserved family among eukaryotes and members have been described in most of the prominent model organisms. The BDCPs were discovered through exploration of a severe human disease comprising symptoms such as albinism, bleeding diathesis, immunodeficiency and cancer growth. On the cellular level, BDCPs are classically known to be involved in membrane trafficking processes including vesicle transport, autophagy as well as membrane fission and fusion events. In accordance, the Arabidopsis thaliana cell morphogenesis and BDCP-coding gene SPIRRIG is implicated in membrane-dependent processes as well. The protein is a particularly interesting BDCP, as to date, it is the only member of this family known to exert an additional function in mRNA metabolism by mediating mRNA stability and promoting processing body formation biologically relevant to A. thaliana salt stress response. The exact molecular mechanism of SPIRRIG, however, remains elusive. This study was set out to investigate SPIRRIG function using two different approaches. One part of this work aimed to gain further understanding of SPIRRIG’s molecular function in salt stress response by analyzing an interplay with tandem zinc finger (TZF) proteins. While molecular functions of plant TZF proteins are not yet fully understood, mammalian homologs are involved in stress-associated mRNA destabilization processes upon binding to specific elements in 3’UTRs of target mRNAs. An interaction of SPIRRIG with TZF10 strongly suggested a potential connection of the two proteins and prompted further investigations in order to unravel this so far unknown interplay between BDCPs and TZFs. Strikingly, this study confirmed TZF10 to rescue the salt hypersensitivity phenotype of spirrig mutants. Extensive mRNA expression and stability assays however could not confirm previously hypothesized, biologically relevant roles of the TZF10 3’UTR in the context of spirrig salt hypersensitivity rescue. Nonetheless, this work convincingly demonstrated general mRNA destabilization capacity mediated by the TZF10 3’UTR and isolated a potential mRNA destabilization-inducing motif. The second part of this work utilized an evolutionarily comparative approach in order to unravel conserved, basal BDCP functions in one of the first plants colonizing the land, the liverwort Marchantia polymorpha. The characterization on the morphological and cellular level of a Mpspirrig mutant, which was identified in a T-DNA screen by its short rhizoids, revealed exciting insights on potential BDCP function in the ancient plant. The Mpspirrig short rhizoid phenotype, reminiscent to the short root hair phenotype of Atspirrig, was surprisingly shown to be an expression of a phenotype hallmarked by an increased number of at the same time significantly smaller cells in Mpspirrig gemmae. This newly discovered phenotype pointed to a role of MpSPIRRIG in cell proliferation and/or cell expansion processes. Moreover, this work strongly supported a connection of MpSPIRRIG to membrane trafficking pathways by demonstrating direct co-localization and interaction of MpSPIRRIG with endosomal sorting complex required for transport (ESCRT) components. Finally, this study revealed MpSPIRRIG to be relevant to Marchantia salt stress response and to locate to cytoplasmic mRNA granules, providing several indications for the dual function of SPIRRIG in the two assumingly unrelated pathways of salt stress response and membrane trafficking to be evolutionarily conserved. |
