Biochemical Characterization and RNA Binding Specificity of LSm Protein SCD6 from the Budding Yeast S. Cerevisiae

The eukaryotic Sm and Sm-like (LSm) proteins form a large family characterized by the presence of an Sm-fold, a structural fold associated with the RNA metabolism. These proteins form homo- or hetero-oligomeric ring consisting of six or seven subunits with a central pore that binds RNA and regulates its biological fates, including splicing, translation, transport, and degradation. This thesis project focuses on an LSm protein, Scd6, from the budding yeast Saccharomyces cerevisiae. In eukaryotic cells, almost 30% proteins fold and mature inside the endoplasmic reticulum (ER). If proteins fail to fold correctly or mis-fold, they accumulate inside the ER, causing a state known as ER stress. In response, cells activate a cellular response known as the unfolded protein response (UPR), a protective mechanism to restore ER protein homeostasis or proteostasis. In yeast S. cerevisiae, UPR is initiated by unconventional splicing of a translationally repressed HAC1 mRNA mediated by an endonuclease Ire1 and a tRNA ligase. The spliced HAC1 mRNA is then translated into Hac1 protein, a transcription factor that induces expression of protein-folding enzyme genes and chaperones, thereby alleviating ER stress. The translational repression in HAC1 mRNA is caused by a secondary structure formed by base-pair interaction between 5’-untraslated region (5’-UTR) and intronic sequences. Research from our collaborator, Dr. Dey at the UW-Milwaukee, has identified that the LSm protein Scd6 plays as a regulatory factor involved in the translational repression of HAC1 mRNA. Scd6 is a conserved protein present in yeast to humans. Little is known about its RNA binding specificity, overall protein architecture, and translational regulation. This master’s dissertation project aims at characterizing the RNA-binding properties and global tertiary structure of Scd6 in vitro, particularly focusing on how Scd6 recognizes a double stranded RNA (dsRNA) found in HAC1 pre-mRNA. The HAC1 pre-mRNA regulatory element and the Scd6 protein serve as an ideal model system to better understand RNA-protein conformational transitions that occur in translation initiation during times of eukaryotic cell stress. Scd6 was successfully overexpressed and purified using affinity chromatography and size-exclusion chromatography (SEC). Biophysical characterization by dynamic light scattering (DLS) suggested that Scd6 may exist in an oligomeric form in solution. Notably, the presence of dsRNA enhanced Scd6 stability over time, supporting its role in targeted RNA–protein interactions. These findings are significant, as LSm domain are traditionally known to bind ssRNA, yet our results demonstrate that Scd6 can recognizes dsRNA, which suggest Scd6 may have a broader role in RNA regulation.