17th Annual Child Health Research Days

Virtual Conference

Oct. 6 & 7, 2021

Abstracts

#47 The Bowen-Conradi Hutterite Syndrome Mutation Reduces Cell Growth and May Alter Ribosome Structure


Trent Nelson, Brandon University; Gol Roberts, Brandon University; Courtney Harris, Brandon University; J. Michael Charette, Brandon University


Introduction

Bowen-Conradi Syndrome (BCS) is a lethal disorder in the Hutterites of the Canadian Prairies. It is due to a D86G mutation in the ribosome assembly/SSU processome protein Emg1 making it a ribosomopathy, a new group of genetic disorders associated with ribosome assembly defects. Emg1 is a pseudouridine methyltransferase that targets the 18S rRNA in the decoding center, a region involved in translational fidelity. Our objective is to create a yeast model of BCS to use in further understanding the essential ribosome assembly protein Emg1 and the molecular consequences of BCS mutant Emg1 in ribosome biogenesis.


Methods

We have created a yeast model of BCS by replacing the endogenous Emg1 promoter with a conditional promoter allowing for depletion of the endogenous protein. The BCS and other mutant and truncated Emg1 constructs were FLAG-tagged and constitutively expressed from a yeast plasmid.


Results

Using growth as a proxy for ribosome assembly, we observed a growth defect in our BCS model, likely due to ribosome mis-assembly, which will be further characterized by pre-rRNA processing northern analysis. Antibiotic sensitivity assays, used to examine structural perturbations to the ribosome, suggest that BCS mutant ribosomes may be structurally and thus translationally compromised, which we will further assay using a series of reporter plasmids. We have also identified a natively unstructured region in the N-terminal domain of Emg1 and determined that it is essential for growth and for the stability of the protein. Lastly, we will monitor incorporation of BCS mutant Emg1 into the SSU processome by protein and RNA co-IPs.


Conclusion

Thus, we have constructed and partially validated a yeast model of BCS, showing that the mutation causes a growth defect and is likely to perturb the structure and thus the translational fidelity of the ribosome.