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-binding͞ 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.

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°C and 32°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°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. ribosome ͉ polysome ͉ hypothermia ͉ RNA-binding protein M ammalian cells exposed to mild hypothermia show a general inhibition of protein synthesis (1, 2) and a concomitant increase in the expression of a small number of cold-shock mRNAs and proteins (3). These proteins appear to protect cells by enabling them to more effectively tolerate cold-stress conditions (reviewed in refs. 3 and 4). In addition, it has been suggested that some cold-shock proteins may have important roles in human testis and skin, where cell temperatures are typically 30-34°C and Ϸ33°C, respectively (3).One of the mRNAs induced by mild hypothermia encodes the RNA binding motif protein 3 (Rbm3) (5-7). This protein is expressed in a wide variety of human fetal tissues (5) and is subjected to both transcriptional and translational regulation in response to mild hypothermia (6, 8). Rbm3 contains a glycinerich carboxyl terminus and can bind to both RNA and DNA via an amino-terminal RNA binding domain (ref. 5 and references therein). This protein is a member of a family of glycine-rich RNA binding proteins that include the cold-inducible RNAbinding protein (CIRP) (6,7,9,10). Although the function of this family of proteins is not yet known, it has been suggested that they affect both transcription and translation (11-13) and that they may facilitate translation at colder temperatures by functioning as RNA c...

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.

This study addresses the properties of a newly identified internal ribosome entry site (IRES) contained within the mRNA of the homeodomain protein Gtx. Sequential deletions of the 5 untranslated region (UTR) from either end did not define distinct IRES boundaries; when five nonoverlapping UTR fragments were tested, four had IRES activity. These observations are consistent with other cellular IRES analyses suggesting that some cellular IRESes are composed of segments (IRES modules) that independently and combinatorially contribute to overall IRES activity. We characterize a 9-nt IRES module from the Gtx 5 UTR that is 100% complementary to the 18S rRNA at nucleotides 1132-1124. In previous work, we demonstrated that this mRNA segment could be crosslinked to its complement within intact 40S subunits. Here we show that increasing the number of copies of this IRES module in the intercistronic region of a dicistronic mRNA strongly enhances IRES activity in various cell lines. Ten linked copies increased IRES activity up to 570-fold in Neuro 2a cells. This level of IRES activity is up to 63-fold greater than that obtained by using the well characterized encephalomyocarditis virus IRES when tested in the same assay system. When the number of nucleotides between two of the 9-nt Gtx IRES modules was increased, the synergy between them decreased. In light of these findings, we discuss possible mechanisms of ribosome recruitment by cellular mRNAs, address the proposed role of higher order RNA structures on cellular IRES activity, and suggest parallels between IRES modules and transcriptional enhancer elements.