Most amino acids may be encoded by more than one codon.  Although the multiple codons that code for the same amino acid all perform the same function, these codons are unequally represented among RNA transcripts, a phenomenon called codon bias.  A closely related concept, codon optimization, is the selection from among multiple codons coding for the same amino acid of the codon(s) most efficiently matched with a corresponding tRNA during translation.  Codon optimization is thought to be one of several factors contributing to codon bias.Codon optimization has been employed in the development of experimental nucleic acid sequences.  Like viruses, which influence host cell gene expression by introducing foreign RNA or DNA into cells, such experimental nucleic acids have been explored in hopes of therapeutically influencing gene expression.Researchers investigated the effects of infection with a specific virus on RNA splicing by inserting a reporter gene into a human-derived cell line and measuring the fluorescence emitted from infected and noninfected cells.  When spliced, the reporter emits fluorescence derived from green fluorescent protein.  In the absence of splicing, no fluorescence is observed.  The effect of viral infection on splicing is shown in Figure 1.Figure 1  Effect of viral infection on splicing (Note:  Bars represent means and standard errors from three experiments and are expressed relative to the mean fluorescence of noninfected cells.)The researchers also tested the influence of codon optimization on expression of a protein encoded by an experimental messenger RNA (mRNA) therapeutic agent.  Codons were optimized by replacing third-position A with G or C when possible and replacing all uridine nucleosides with N1-methyl-pseudouridine (N1-methyl-Ψ).  Pseudouridine (Ψ) is a naturally occurring, post-transcriptionally modified uridine.  Base pairing of selected nucleotides is shown in Table 1.  Researchers concluded that codon optimization increased translational efficiency, based on a ratio of measured protein to mRNA amounts of 0.1 without optimization and 0.4 with optimization.Table 1  Base Pairing of Selected NucleotidesHia, F., Yang, S. F., Shichino, Y., Yoshinaga, M., Murakawa, Y., Vandenbon, A., Fukao, A., Fujiwara, T., Landthaler, M., Natsume, T., Adachi, S., Iwasaki, S., & Takeuchi, O. (2019). Codon bias confers stability to human mRNAs. EMBO Reports, 20(11), e48220. Question 42Based on the passage, infection with the virus studied by the researchers likely:increases the joining of introns separated by an exon.decreases the joining of exons separated by an intron.increases the number of different protein isoforms expressed.A.I onlyB.II onlyC.III onlyD.I and II onlySubmit