Vincent P. Mauro

Physical evidence supporting a ribosomal shunting mechanism of translation initiation for BACE1 mRNA.

In Alzheimer disease, elevated levels of the BACE1 enzyme are correlated with increased production of amyloid peptides and disease pathology. The increase in BACE1 levels is post-transcriptional and may involve altered translation efficiency. Earlier studies have indicated that translation of BACE1 mRNA is cap-dependent. As ribosomal subunits move from the cap-structure to the initiation codon, they fail to recognize several AUG codons in the 5′ leader. In this study, we looked for physical evidence of the mechanism underlying ribosomal scanning or shunting along the BACE1 5′ leader by investigating structural stability in the 5′ leaders of endogenous mRNAs in vivo. To perform this analysis, we probed RNAs using lead(II) acetate, a cell-permeable chemical that induces cleavage of unpaired nucleotides having conformational flexibility. The data revealed that the ≈440-nt 5′ leader was generally resistant to cleavage except for a region upstream of the initiation codon. Cleavage continued into the coding region, consistent with destabilization of secondary structures by translating ribosomes. Evidence that a large segment of the BACE1 5′ leader was not cleaved indicates that this region is structurally stable and suggests that it is not scanned. The data support a mechanism of translation initiation in which ribosomal subunits bypass (shunt) part of the BACE1 5′ leader to reach the initiation codon. We suggest that a nucleotide bias in the 5′ leader may predispose the initiation codon to be more accessible than other AUG codons in the 5′ leader, leading to an increase in its relative utilization.

Considerations in the Use of Codon Optimization for Recombinant Protein Expression

Codon optimization is a gene engineering approach that is commonly used for enhancing recombinant protein expression. This approach is possible because (1) degeneracy of the genetic code enables most amino acids to be encoded by multiple codons and (2) different mRNAs encoding the same protein can vary dramatically in the amount of protein expressed. However, because codon optimization potentially disrupts overlapping information encoded in mRNA coding regions, protein structure and function may be altered. This chapter discusses the use of codon optimization for various applications in mammalian cells as well as potential consequences, so that informed decisions can be made on the appropriateness of using this approach in each case.