Non-Optimal Codon Usage Shapes Cell Cycle Regulators: Codon Optimization Studies on CDK1 and NUF2

The expression and functions of genes are largely dependent on genome integrity and stability. Codon usage plays a significant role in maintaining stability and functions of genes. Perturbation in codon sequence can lead to functional and structural dysfunction of a gene. In our study, we performed the codon usage analyses of cell cycle-dependent genes. Various codon usage parameters were analyzed using nucleotide compositions like RSCU, ENC, and GC analyses. Codon usage analysis using nucleotide composition analysis showed that cell cycle dependent genes follow non-optimal codon usage. Genes preferred AT-rich ending over GC-rich ending, thereby suggesting preference for non-optimal codons. Neutrality and parity plot showed that the codon preference is a result of mutation selection pressure. To study the codon usage implications, we optimized the codons of cell cycle dependent genes (CDK1 and NUF2) to study their effects on cell cycle as well as on the apoptosis in vitro. We observed that codon optimization alters the cell cycle length in cell cycle-dependent genes. Codon changes further influence cell fate and survival, highlighting the impact of codon usage on cellular outcomes. Apart from affecting the functional aspects of the proteins, our studies revealed that codon usage preferences directly affect the stability of both mRNA and proteins. Specifically, genes and proteins with non-optimal codons exhibited reduced stability compared to their optimized counterparts, suggesting critical implications for cell cycle regulation and apoptosis.