A scalable genetic tool for the functional analysis of the signal recognition particle

Abstract

AbstractMutations in theSRP54gene are linked to the pathophysiology of severe congenital neutropenia (SCN).SRP54is a key protein comprising one of the six protein subunits of the signal recognition particle responsible for co-translational targeting of proteins to the ER; mutations inSRP54disrupt this process. Crystal structures and biochemical characterization of a fewSRP54mutants provide insights into howSRP54mutations affect its function. However, to date, no scalable, flexible platform exists to study the sequence-structure-function relationships of SRP54 mutations and perform functional genomics and genome-wide association studies. In this work, we established a haploid model inSaccharomyces cerevisiaebased on inducible gene expression that allows these relationships to be studied. We employed this model to test the function of orthologous clinical mutations to demonstrate the model’s suitability for studying SCN. Lastly, we demonstrate the suspected dominant-negative phenotypes associated withSRP54mutants. In doing so, we discovered for the first time that the most common yeast orthologous clinical mutation, S125del (T117del human orthologue) displayed the least severe growth defect while the less common G234E mutant (G226E human orthologue) displayed the most severe growth defect. The ability of this haploid model to recapitulate these phenotypes while remaining amenable to high-throughput screening approaches makes it a powerful tool for studyingSRP54. Furthermore, the methodology used to create this model may also be used to study other human diseases involving essential and quasi-essential genes.TakeawayThe constitutively expressed quasi-essential nativeSRP54gene in yeast is replaced with an aldosterone-inducible version, making a yeast haploid strain, LFL001, that requires aldosterone for growth.Strains developed in this work corroborated that yeastSRP54mutations exhibit dominant-negative phenotypes.The LFL000 and LFL001 yeast strains developed here are uniquely positioned to interrogate sequence-structure-function relationships ofSRP54.