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 model’s 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.