Characterization of AGO3 Variants of Unknown Significance: Impact on Biophysical Properties and the Transcriptome

Author

Shala Baxton Munn IIFollow

Date of Award

12-2023

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Genetics and Biochemistry

Committee Chair/Advisor

Lela Lackey

Committee Member

Robert Anholt

Committee Member

Heather Flanagan-Steet

Committee Member

Liangjiang Wang

Abstract

The four human Argonaute family members (AGO1-4) are involved in gene regulation. All four proteins complex with small RNAs to form RNA-Induced Silencing Complexes (RISCs). Many studies have documented the ability of AGO2 to regulate gene expression, including through endonucleolytic cleavage of transcripts. The Argonaute protein AGO3, however, is poorly understood. Only a handful of studies have focused on AGO3 activity and provide minimal support for RNA silencing functionality. However, combined microdeletions in AGO1 and AGO3 are associated with dysmorphic features, poor feeding habits, and developmental delays. Recently, two patient variants in the coding sequence of AGO3 (Patient 1 – R507W & Patient 2 – F225L) were reported in patients with similar symptoms. Neither variant of unknown significance (VUS) can be properly characterized without understanding AGO3 function. We sought to characterize the molecular impact of the patient variants (P1 & P2), in addition to a catalytic variant, E638A, to better understand AGO3 function. We utilized a combination of stability and structure predictions, literature review on AGO3, RNA-seq, and RNA structure probing to elucidate the effects of the variants. The patient variants did not significantly alter the predicted stability or structure of AGO3, even though AGO3 was expected to be intolerant of such variants and the variants were conserved amongst related species (shared identity of 98.9%). RNA sequencing analysis from patient fibroblast cells containing the variants of interest identified differential gene expression, particularly in P2. Gene ontology of differentially expressed genes in P2 revealed an enrichment in cell morphology pathways, including cell and neuron projection pathways. Targeting these pathways were a set of miRNAs, which were enriched for these genes compared to randomized genes. To verify the fibroblast sequencing, we attempted to create AGO3 polyclonal cell lines but failed to properly express the protein or RNA. Lastly, we found that the P2 variant also caused structural changes the coding region of AGO3 mRNA. Altogether, our results suggest that the AGO3 P2 variant can disrupt gene networks and AGO3 RNA structure. Additional research is necessary to fully understand the biophysical and transcriptome-wide effects of AGO3 variants and their impact on cellular and organismal biology.

Recommended Citation

Munn, Shala Baxton II, "Characterization of AGO3 Variants of Unknown Significance: Impact on Biophysical Properties and the Transcriptome" (2023). All Theses. 4188.
https://tigerprints.clemson.edu/all_theses/4188