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

Abstract

Mutations in the SRP54 gene are linked to the pathophysiology of severe congenital neutropenia (SCN). SRP54 is 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 in SRP54 disrupt this process. Crystal structures and biochemical characterization of a few SRP54 mutants provide insights into how SRP54 mutations 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 in Saccharomyces cerevisiae based 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 models suitability for studying SCN. Lastly, we demonstrate the suspected dominant-negative phenotypes associated with SRP54 mutants. 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 studying SRP54. Furthermore, the methodology used to create this model may also be used to study other human diseases involving essential and quasi-essential genes. TakeawayO_LIThe constitutively expressed quasi-essential native SRP54 gene in yeast is replaced with an aldosterone-inducible version, making a yeast haploid strain, LFL001, that requires aldosterone for growth. C_LIO_LIStrains developed in this work corroborated that yeast SRP54 mutations exhibit dominant-negative phenotypes. C_LIO_LIThe LFL000 and LFL001 yeast strains developed here are uniquely positioned to interrogate sequence-structure-function relationships of SRP54. C_LI