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.

Cell-free protein synthesis has emerged as a powerful technology for rapid and efficient protein production. Cell-free methods are also amenable to automation and such systems have been extensively used for high-throughput protein production and screening; however, current fluidic systems are not adequate for manufacturing protein biopharmaceuticals. In this work, we report on the initial development of a fluidic process for rapid end-to-end production of recombinant protein biologics. This process incorporates a bioreactor module that can be used with eukaryotic or prokaryotic lysates that are programmed for combined transcription/translation of an engineered DNA template encoding for specific protein targets. Purification of the cell-free expressed product occurs through a series of protein separation modules that are configurable for process-specific isolation of different proteins. Using this approach, we demonstrate production of two bioactive human protein therapeutics, erythropoietin and granulocyte-macrophage colony-stimulating factor, in yeast and bacterial extracts, respectively, each within 24 hours. This process is flexible, scalable and amenable to automation for rapid production at the point-of-need of proteins with significant pharmaceutical, medical, or biotechnological value.

Codon optimization describes gene engineering approaches that use synonymous codon changes to increase protein production. Applications for codon optimization include recombinant protein drugs and nucleic acid therapies, including gene therapy, mRNA therapy, and DNA/RNA vaccines. However, recent reports indicate that codon optimization can affect protein conformation and function, increase immunogenicity, and reduce efficacy. We critically review this subject, identifying additional potential hazards including some unique to nucleic acid therapies. This analysis highlights the evolved complexity of codon usage and challenges the scientific bases for codon optimization. Consequently, codon optimization may not provide the optimal strategy for increasing protein production and may decrease the safety and efficacy of biotech therapeutics. We suggest that the use of this approach is reconsidered, particularly for in vivo applications.

In eukaryotes, translation initiation involves recruitment of ribosomal subunits at either the 5' m7G cap structure or at an internal ribosome entry site (IRES). For most mRNAs, the initiation codon is located some distance downstream, necessitating ribosomal movement to this site. Although the mechanistic details of this movement remain to be fully resolved, it appears to be nonlinear for some mRNAs (i.e., ribosomal subunits appear to bypass [shunt] segments of the 5' leader as they move to the initiation codon). This chapter describes various experimental approaches to assess ribosomal shunting and to identify mRNA elements (shunt sites) that facilitate shunting. In addition, we provide an overview of approaches that can be used to investigate the mechanism used by individual shunt sites, along with a detailed protocol for investigating putative base pairing interactions between shunt sites and 18S rRNA.

In eukaryotes, 40S ribosomal subunits move from their recruitment site on the mRNA to the initiation codon by an as yet poorly understood process. One postulated mechanism involves ribosomal shunting, in which ribosomal subunits completely bypass regions of the 5' leader. For some mRNAs, shunting has been shown to require various mRNA elements, some of which are thought to base pair to 18S rRNA; however, the role of base pairing has not yet been tested directly. In earlier studies, we demonstrated that a short mRNA element in the 5' leader of the Gtx homeodomain mRNA functioned as a ribosomal recruitment site by base pairing to the 18S rRNA. Using a model system to assess translation in transfected cells, we now show that this intermolecular interaction also facilitates ribosomal shunting across two types of obstacles: an upstream AUG codon in excellent context or a stable hairpin structure. Highly efficient shunting occurred when multiple Gtx elements were present upstream of the obstacles, and a single Gtx element was present downstream. Shunting was less efficient, however, when the multiple Gtx elements were present only upstream of the obstacles. In addition, control experiments with mRNAs lacking the upstream elements showed that these results could not be attributed to recruitment by the single downstream element. Experiments in yeast in which the mRNA elements and 18S rRNA sequences were both mutated indicated that shunting required an intact complementary match. The data obtained by this model system provide direct evidence that ribosomal shunting can be mediated by mRNA-rRNA base pairing, a finding that may have general implications for mechanisms of ribosome movement.

Eukaryotic mRNAs often recruit ribosomal subunits some distance upstream of the initiation codon; however, the mechanisms by which they reach the initiation codon remain to be fully elucidated. Although scanning is a widely accepted model, evidence for alternative mechanisms has accumulated. We previously suggested that this process may involve tethering of ribosomal complexes to the mRNA, in which the intervening mRNA is bypassed, or clustering, in which the initiation codon is reached by dynamic binding and release of ribosomal subunits at internal sites. The present studies tested the feasibility of these ideas by using model mRNAs and revealed that translation efficiency varied with the distance between the site of ribosomal recruitment and the initiation codon. The present studies also showed that translation could initiate efficiently at AUG codons located upstream of an internal site. These observations are consistent with ribosomal tethering at the cap structure and clustering at internal sites.

Base-pairing of messenger RNA to ribosomal RNA is a mechanism of translation initiation in prokaryotes. Although analogous base-pairing has been suggested to affect the translation of various eukaryotic mRNAs, direct evidence has been lacking. To test such base-pairing, we developed a yeast system that uses ribosomes containing a mouse-yeast hybrid 18S rRNA. Using this system, we demonstrate that a 9-nucleotide element found in the mouse Gtx homeodomain mRNA facilitates translation initiation by base-pairing to 18S rRNA. Various point mutations in the Gtx element and in either the hybrid or wild-type yeast 18S rRNAs confirmed the requirement for an intact complementary match. The presence of the Gtx element in various mRNAs suggests that this element affects the translation of groups of mRNAs. We discuss the possibility that other mRNA elements affect translation by base-pairing to different sites in the 18S rRNA.

We previously identified an internal ribosome entry site (IRES) within the 5' leader of the mRNA encoding the Gtx homeodomain protein and showed that shorter nonoverlapping segments of this 5' leader could enhance the translation of a second cistron in a dicistronic mRNA. One of these segments was 9 nt in length, and when multiple copies of this IRES module were linked together, IRES activity was greatly enhanced. To further expand the potential uses of these synthetic constructs and facilitate analyses of the mechanism by which they affect translation, we show here that an IRES containing five linked copies of the 9-nt sequence can also enhance translation in the 5' leader of a monocistronic mRNA. Moreover, a search for interactions of the IRES module with cellular factors revealed specific binding to 40S ribosomal subunits but not to other cellular components. Based on the results of earlier studies suggesting that this sequence could bind to a complementary segment of 18S rRNA, we tested various sequences for possible links between the length of the complementary match, their binding to ribosomes, and their influence on translational efficiency. We found that the length of the complementary match was directly correlated with the ability of RNA probes to bind to ribosomes. In addition, translation was maximally enhanced ( approximately 8-fold) by a 7-nt segment of the 9-nt element; the enhancement declined progressively as the complementary stretches became progressively longer or shorter. The results suggest that the Gtx 9-nt sequence affects translation efficiency by a mechanism that involves base pairing to 18S rRNA.

Although the internal ribosome entry sites (IRESes) of viral mRNAs are highly structured and comprise several hundred nucleotides, there is a variety of evidence indicating that very short nucleotide sequences, both naturally occurring and synthetic, can similarly mediate internal initiation of translation. In this study, we performed deletion and mutational analyses of an IRES contained within the 720-nucleotide (nt) 5' leader of the Rbm3 mRNA and demonstrated that this IRES is highly modular, with at least 9 discrete cis-acting sequences. These cis-acting sequences include a 22-nt IRES module, a 10-nt enhancer, and 2 inhibitory sequences. The 22-nt sequence was shown to function as an IRES when tested in isolation, and we demonstrated that it did not enhance translation by functioning as a transcriptional promoter, enhancer, or splice site. The activities of all 4 cis-acting sequences were further confirmed by their mutation in the context of the full IRES. Interestingly, one of the inhibitory cis-acting sequences is contained within an upstream open reading frame (uORF), and its activity seems to be masked by translation of this uORF. Binding studies revealed that all 4 cis-acting sequences could bind specifically to distinct cytoplasmic proteins. In addition, the 22-nt IRES module was shown to bind specifically to 40 S ribosomal subunits. The results demonstrate that different types of cis-acting sequences mediate or modulate translation of the Rbm3 mRNA and suggest that one of the IRES modules contained within the 5' leader facilitates translation initiation by binding directly to 40 S ribosomal subunits.

The 5′ untranslated region of the proto-oncogene c- myc contains an internal ribosome entry segment (IRES) ( Nanbru et al., 1997 ; Stoneley et al., 1998 ) and thus c- myc protein synthesis can be initiated by a cap-independent as well as a cap-dependent mechanism ( Stoneley et al., 2000 ). In cell lines derived from patients with multiple myeloma (MM) there is aberrant translational regulation of c- myc and this correlates with a C-T mutation in the c- myc -IRES ( Paulin et al., 1996 ). RNA derived from the mutant IRES displays enhanced binding of protein factors ( Paulin et al., 1998 ). Here we show that the same mutation is present in 42% of bone marrow samples obtained from patients with MM, but was not present in any of 21 controls demonstrating a strong correlation between this mutation and the disease. In a tissue culture based assay, the mutant version of the c- myc -IRES was more active in all cell types tested, but showed the greatest activity in a cell line derived from a patient with MM. Our data demonstrate that a single mutation in the c- myc -IRES is sufficient to cause enhanced initiation of translation via internal ribosome entry and represents a novel mechanism of oncogenesis.

Translation in eukaryotic cells is generally initiated by ribosome scanning from the 5' end of the capped mRNA. However, initiation of translation may also occur by a mechanism which is independent of the cap structure and in this case ribosomes are directed to the start codon by an internal ribosome entry segment (IRES). Picornaviruses represent the paradigm for this mechanism, but only a few examples exist which show that this mechanism is used by eukaryotic cells. In this report we show data which demonstrate that the 5' UTR of the proto-oncogene c-myc contains an IRES. When a dicistronic reporter vector, with c-myc 5' UTR inserted between the two cistrons, was transfected into both HepG2 and HeLa cells, the translation of the downstream cistron was increased by 50-fold, demonstrating that translational regulation of c-myc is mediated through cap-independent mechanisms. This is the first example of a proto-oncogene regulated in this manner and suggests that aberrant translational regulation of c-myc is likely to play a role in tumorigenesis.

A 340 nucleotide section of the c-myc 5' untranslated region (UTR) contains an internal ribosome entry segment. We have described previously a mutation in this region of RNA in cell lines derived from patients with multiple myeloma (MM) which exhibit increased expression of c-myc protein by an aberrant translational mechanism. In this study we show by electrophoretic mobility shift assays (EMSA), north-western blotting and UV cross-linking that radiolabelled c-myc 5' UTR RNA transcripts which harbour the mutation-cause enhanced binding of cellular proteins. In addition, we also demonstrate that an MM derived cell line possesses an altered repertoire of RNA binding proteins. Our data suggest that the deregulated expression of c-myc in MM could result both from the effect of the mutation and the additional proteins which are-present in these cell types.