Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo.

Degeneracy in eukaryotic translation initiation is evident in the initiation strategies of various viruses. Hepatitis C virus (HCV) provides an exceptional example--translation of the HCV RNA is facilitated by an internal ribosome entry site (IRES) that can autonomously bind a 40S ribosomal subunit and accurately position it at the initiation codon. This binding involves both ribosomal protein and 18S ribosomal RNA (rRNA) interactions. In this study, we evaluate the functional significance of the rRNA interaction and show that HCV IRES activity requires a 3-nt Watson-Crick base-pairing interaction between the apical loop of subdomain IIId in the IRES and helix 26 in 18S rRNA. Mutations of these nucleotides in either RNA dramatically disrupted IRES activity. The activities of the mutated HCV IRESs could be restored by compensatory mutations in the 18S rRNA. The effects of the 18S rRNA mutations appeared to be specific inasmuch as ribosomes containing these mutations did not support translation mediated by the wild-type HCV IRES, but did not block translation mediated by the cap structure or other viral IRESs. The present study provides, to our knowledge, the first functional demonstration of mRNA-rRNA base pairing in mammalian cells. By contrast with other rRNA-binding sites in mRNAs that can enhance translation as independent elements, e.g., the Shine-Dalgarno sequence in prokaryotes, the rRNA-binding site in the HCV IRES functions as an essential component of a more complex interaction.

Translation regulation by ribosomes: Increased complexity and expanded scope.

The primary function of ribosomes is to decode mRNAs into polypeptide chains; however, this description is overly simplistic. Accumulating evidence shows that ribosomes themselves can affect the relative efficiency with which various mRNAs are translated and indicates that these effects can be modulated by ribosome heterogeneity. The notion that ribosomes have regulatory capabilities was elaborated more than a decade ago in the ribosome filter hypothesis. Various lines of evidence support this idea and have shown that the translation of some mRNAs is affected by discrete binding interactions with rRNA or ribosomal proteins. Recent work from our laboratory has demonstrated that base-pairing of the Hepatitis C Virus (HCV) internal ribosome entry site (IRES) to 18S rRNA is required for IRES function, but only in the context of more complex ribosomal interactions. The HCV IRES provides an example of the ribosome filter that involves multiple binding interactions between mRNAs and ribosomal subunits.

Analysis of rRNA processing and translation in mammalian cells using a synthetic 18S rRNA expression system.

Analysis of processing, assembly, and function of higher eukaryotic ribosomal RNA (rRNA) has been hindered by the lack of an expression system that enables rRNA to be modified and then examined functionally. Given the potential usefulness of such a system, we have developed one for mammalian 18S rRNA. We inserted a sequence tag into expansion segment 3 of mouse 18S rRNA to monitor expression and cleavage by hybridization. Mutations were identified that confer resistance to pactamycin, allowing functional analysis of 40S ribosomal subunits containing synthetic 18S rRNAs by selectively blocking translation from endogenous (pactamycin-sensitive) subunits. rRNA constructs were suitably expressed in transfected cells, shown to process correctly, incorporate into ≈ 15% of 40S subunits, and function normally based on various criteria. After rigorous analysis, the system was used to investigate the importance of sequences that flank 18S rRNA in precursor transcripts. Although deletion analysis supported the requirement of binding sites for the U3 snoRNA, it showed that a large segment of the 5' external transcribed spacer and the entire first internal transcribed spacer, both of which flank 18S rRNA, are not required. The success of this approach opens the possibility of functional analyses of ribosomes, with applications in basic research and synthetic biology.

An mRNA-rRNA base-pairing mechanism for translation initiation in eukaryotes.

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.

Reconciling contradictory reports regarding translation of BACE1 mRNA: initiation mechanism is altered by different expression systems.

We previously showed that translation from the rat BACE1 5' leader is cap-dependent and that four AUG codons (AUG1-4) in the 5' leader were bypassed, partially or completely, depending on the cell line. Two other groups reported comparable results with human BACE1 sequences in different cell lines, although different mechanisms were postulated. In contrast, a third group working with the human sequence reported that most translation events are initiated at AUG2. Using reporter constructs with the rat BACE1 5' leader in rat cells, we now show that this apparent discrepancy between studies can be explained by the use of different expression systems and differences in interpretation. When reporter constructs were transcribed in the nucleus, the upstream AUG codons did not affect translation, but when mRNAs were transcribed in the cytoplasm or when in vitro transcripts were transfected into cells, the upstream AUG codons inhibited translation. These findings suggest that when transcription occurs in the nucleus, the BACE1 mRNA initiates translation by a shunting mechanism. The results are less consistent with either leaky scanning or reinitiation and provide a caveat against the use of cytoplasmic expression systems or RNA transfection for analyses of translation initiation.

Codon Optimization in the Production of Recombinant Biotherapeutics: Potential Risks and Considerations.

Biotherapeutics are increasingly becoming the mainstay in the treatment of a variety of human conditions, particularly in oncology and hematology. The production of therapeutic antibodies, cytokines, and fusion proteins have markedly accelerated these fields over the past decade and are probably the major contributor to improved patient outcomes. Today, most protein therapeutics are expressed as recombinant proteins in mammalian cell lines. An expression technology commonly used to increase protein levels involves codon optimization. This approach is possible because degeneracy of the genetic code enables most amino acids to be encoded by more than one synonymous codon and because codon usage can have a pronounced influence on levels of protein expression. Indeed, codon optimization has been reported to increase protein expression by >  1000-fold. The primary tactic of codon optimization is to increase the rate of translation elongation by overcoming limitations associated with species-specific differences in codon usage and transfer RNA (tRNA) abundance. However, in mammalian cells, assumptions underlying codon optimization appear to be poorly supported or unfounded. Moreover, because not all synonymous codon mutations are neutral, codon optimization can lead to alterations in protein conformation and function. This review discusses codon optimization for therapeutic protein production in mammalian cells.

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.

Analysis of ribosomal shunting during translation initiation in eukaryotic mRNAs.

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.

A cell-free expression and purification process for rapid production of protein biologics.

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.

A critical analysis of codon optimization in human therapeutics.

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.

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.

Determinants of initiation codon selection during translation in mammalian cells.

Factors affecting translation of mRNA contribute to the complexity of eukaryotic proteomes. In some cases, translation of a particular mRNA can generate multiple proteins. However, the factors that determine whether ribosomes initiate translation from the first AUG codon in the transcript, from a downstream codon, or from multiple sites are not completely understood. Various mRNA properties, including AUG codon-accessibility and 5' leader length have been proposed as potential determinants that affect where ribosomes initiate translation. To explore this issue, we performed studies using synthetic mRNAs with two in-frame AUG codons-both in excellent context. Open reading frames initiating at AUG1 and AUG2 encode large and small isoforms of a reporter protein, respectively. Translation of such an mRNA in COS-7 cells was shown to be 5' cap-dependent and to occur efficiently from both AUG codons. AUG codon-accessibility was modified by using two different elements: an antisense locked nucleic acid oligonucleotide and an exon-junction complex. When either element was used to mask AUG1, the ratio of the proteins synthesized changed, favoring the smaller (AUG2-initiated) protein. In addition, we observed that increased leader length by itself changed the ratio of the proteins and favored initiation at AUG1. These observations demonstrate that initiation codon selection is affected by various factors, including AUG codon-accessibility and 5' leader length, and is not necessarily determined by the order of AUG codons (5'→3'). The modulation of AUG codon accessibility may provide a powerful means of translation regulation in eukaryotic cells.

Antisense masking reveals contributions of mRNA-rRNA base pairing to translation of Gtx and FGF2 mRNAs.

We previously showed that a 9-nucleotide sequence from the 5' leader of the Gtx homeodomain mRNA facilitates translation initiation by base pairing to 18S rRNA. These earlier studies tested the Gtx element in isolation; we now assess the physiological relevance of this element in the context of two natural mRNAs that contain this sequence in their 5' leaders, Gtx itself and FGF2 (fibroblast growth factor 2). 2'-O-Methyl-modified RNA oligonucleotides were employed to block mRNA-rRNA base pairing by targeting either the Gtx-binding site in 18S rRNA or Gtx elements in recombinant mRNAs containing the Gtx or FGF2 5' leaders linked to a reporter cistron. Studies in cell-free lysates and transfected COS-7 cells showed that translation of mRNAs containing the Gtx or FGF2 5' leaders was decreased by > 50% when oligonucleotides targeting either the rRNA or mRNA were used. Specificity was demonstrated by showing that translation of the recombinant mRNAs was unaffected by control oligonucleotides. In addition, the specific oligonucleotides did not affect the translation of recombinant mRNAs in which the Gtx elements were mutated. Experiments performed using constructs containing Gtx and FGF2 5' leader and coding sequences ruled out possible effects of the reporter cistron. Furthermore, two-dimensional gel electrophoresis revealed that the oligonucleotides used in this study had little overall effect on the proteomes of cells transfected with these oligonucleotides. This study demonstrates that mRNA-rRNA base pairing affects the expression of two cellular mRNAs and describes a new approach for investigating putative mRNA-rRNA base pairing interactions in mammalian cells.

The ribosome filter redux.

The ribosome filter hypothesis postulates that ribosomes are not simply translation machines but also function as regulatory elements that differentially affect or filter the translation of particular mRNAs. On the basis of new information, we take the opportunity here to review the ribosome filter hypothesis, suggest specific mechanisms of action, and discuss recent examples from the literature that support it.

Ribosomal tethering and clustering as mechanisms for translation initiation.

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.

Ribosomal shunting mediated by a translational enhancer element that base pairs to 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.

A positive feedback vector for identification of nucleotide sequences that enhance translation.

In earlier studies, we identified short (6- to 22-nt) sequences that functioned as internal ribosome entry sites (IRESes) and enhanced translation. The size of these IRES elements suggested that they might be prevalent within the messenger population and that individual elements might affect the translation of different groups of mRNAs. To begin to assess the number of different IRES elements in mammalian cells, we have developed a powerful method that uses a positive feedback mechanism to amplify the activities of individual IRES elements. This method uses a vector that encodes a dicistronic mRNA with a reporter gene (Renilla luciferase or the EGFP) as the first cistron and the yeast Gal4/viral protein 16 (VP16) transcription factor as the second cistron. Transcription of this mRNA is driven by a minimal promoter containing four copies of the Gal4 upstream activation sequence. In this method, the presence of an IRES in the intercistronic region facilitates the translation of Gal4/VP16, which binds to the upstream activation sequences and triggers a positive feedback loop that escalates the production of dicistronic mRNA and Gal4/VP16. A corresponding increase in the translation of the first cistron (luciferase or EGFP) is monitored either by measuring luciferase activity or by using FACS. The latter enables IRES-positive cells to be isolated. We present tests of the feedback mechanism by using an IRES module from Gtx homeodomain mRNA and an IRES from hepatitis C virus and demonstrate the utility of this vector system for the screening, identification, and analysis of IRES elements.

Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis.

The expression of Rbm3, a glycine-rich RNA-binding protein, is enhanced under conditions of mild hypothermia, and Rbm3 has been postulated to facilitate protein synthesis at colder temperatures. To investigate this possibility, Rbm3 was overexpressed as a c-Myc fusion protein in mouse neuroblastoma N2a cells. Cells expressing this fusion protein showed a 3-fold increase in protein synthesis at both 37 degrees C and 32 degrees C compared with control cells. Although polysome profiles of cells expressing the fusion protein and control cells were similar, several differences were noted, suggesting that Rbm3 might enhance the association of 40S and 60S ribosomal subunits at 32 degrees C. Studies to assess a direct interaction of Rbm3 with ribosomes showed that a fraction of Rbm3 was associated with 60S ribosomal subunits in an RNA-independent manner. It appeared unlikely that this association could explain the global enhancement of protein synthesis, however, because cells expressing the Rbm3 fusion protein showed no substantial increase in the size of their monosome and polysome peaks, suggesting that similar numbers of mRNAs were being translated at approximately the same rates. In contrast, a complex that sedimented between the top of the gradient and 40S subunits was less abundant in cells expressing recombinant Rbm3. Further analysis showed that the RNA component of this fraction was microRNA. We discuss the possibility that Rbm3 expression alters global protein synthesis by affecting microRNA levels and suggest that both Rbm3 and microRNAs are part of a homeostatic mechanism that regulates global levels of protein synthesis under normal and cold-stress conditions.

The internal ribosome entry site (IRES) contained within the RNA-binding motif protein 3 (Rbm3) mRNA is composed of functionally distinct elements.

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 ribosome filter hypothesis.

A variety of posttranscriptional mechanisms affects the processing, subcellular localization, and translation of messenger RNAs (mRNAs). Translational control appears to occur primarily at the initiation rather than the elongation stage. It has been suggested that translation is mediated largely by means of a cap-binding/scanning mechanism. On the basis of recent findings, we propose here that differential binding of particular mRNAs to eukaryotic 40S ribosomal subunits before translation may also selectively affect rates of polypeptide chain production. In this view, ribosomal subunits themselves are considered to be regulatory elements or filters that mediate interactions between particular mRNAs and components of the translation machinery. Differences in these interactions affect how efficiently individual mRNAs compete for ribosomal subunits. These competitive interactions would depend in part on the complementarity between sequences in mRNA and rRNA, as well as on structural differences among ribosomes in different cell types. By these means, translation may either be enhanced through increased recruitment of ribosomes or inhibited through strong interactions that sequester mRNAs. We propose that ribosomal filters may be important in cell differentiation and describe experimental tests for the filter hypothesis.