The many routes to regulating mRNA translation

A report on the meeting 'Translational Control' at Cold Spring Harbor, New York, 6-10 September 2006.     

The many routes to regulating mRNA translation

A report on the meeting 'Translational Control' at Cold Spring Harbor, New York, 6-10 September 2006.     

mRNA trafficking and local translation: the Yin and Yang of regulating mRNA localization in neurons

Localized translation and the requisite trafficking of the mRNA template play significant roles in the nervous system including the establishment of dendrites and axons, axon path-finding, and synaptic plasticity. We provide a brief review on the regulation of localizing mRNA in mammalian neurons through critical post-translational modifications of the factors involved. These examples highlight the relationship between mRNA trafficking and the translational regulation of trafficked mRNAs and provide insight into how extracellular signals target these events during signal transduction.     

A universal strategy for regulating mRNA translation in prokaryotic and eukaryotic cells

We describe a simple strategy to control mRNA translation in both prokaryotic and eukaryotic cells which relies on a unique protein–RNA interaction. Specifically, we used the Pumilio/FBF (PUF) protein to repress translation by binding in between the ribosome binding site (RBS) and the start codon (in Escherichia coli), or by binding to the 5′ untranslated region of target mRNAs (in mammalian cells). The design principle is straightforward, the extent of translational repression can be tuned and the regulator is genetically encoded, enabling the construction of artificial signal cascades. We demonstrate that this approach can also be used to regulate polycistronic mRNAs; such regulation has rarely been achieved in previous reports. Since the regulator used in this study is a modular RNA-binding protein, which can be engineered to target different 8-nucleotide RNA sequences, our strategy could be used in the future to target endogenous mRNAs for regulating metabolic flows and signaling pathways in both prokaryotic and eukaryotic cells.     

Preparation of pancreatic mRNA: cell-free translation of an insulin-immunoreactive polypeptide.

Total nucleic acid extraction and selection of poly A-containing molecules yield preparative quantities of undegraded mRNA from adult and fetal pancreas. Using a stringent immunoassay, this mRNA is found to direct the synthesis of an immunoreactive insulin polypeptide in the wheat germ translation system. On sodium dodecyl sulfate polyacrylamide gels, this polypeptide (12,000-13,000 daltons) is larger than proinsulin (9,000 daltons).     

Control mechanisms regulating gene expression during normal differentiation of myeloid leukemic cells: Differentiation defective mutants blocked in mRNA production and mRNA translation

Regulation of gene expression during myeloid cell differentiation has been analyzed using clones of myeloid leukemic cells that differ in their competence to be induced to differentiate by the normal macrophage- and granulocyte-inducing protein MGI. Changes in the relative rate of synthesis for specific proteins were compared to changes in the relative amounts of corresponding translatable poly(A)⁺ mRNAs, assayed in the reticulocyte cell-free translation system, using two-dimensional gel electrophoresis. Of the 217 proteins which changed during MGI-induced differentiation of normally differentiating MGI⁺D⁺ leukemic cells, 136 could be identified as products of cell-free translation. Eighty-four percent of the 70 decreases in synthesis, most of which occurred early during differentiation, were not accompanied by a parallel decrease in the amount of translatable mRNA, but were accompanied by a parallel shift of the corresponding mRNAs from the polysomal to the monosomal and free mRNA fractions. These results indicate that most of the early decreases in the synthesis of proteins were translationally regulated. In contrast, 81% of the proteins which increased in synthesis and 71% of the proteins that were induced de novo were regulated at the level of mRNA production. Experiments with differentiation defective mutants have shown that they were blocked both at the level of mRNA production and mRNA translation. The data with these mutants have suggested that there were different subsets of translationally regulated proteins which were separately regulated. The translational blocks for several proteins in these mutant clones have also made it possible to identify additional translational sites of regulation for protein changes that were controlled at the level of mRNA production during normal differentiation. The results indicate that translational regulation may predominantly have a different function in cell differentiation than regulation by mRNA production, and that differentiation-defective mutants can be blocked at either level.     

Axonal mRNA translation: an unexpected link to axon survival and the mitochondrion

Localized mRNA translation plays roles in dendrites and axons, but the regulatory mechanisms and downstream pathways are not well understood. An article in Cell by Yoon et al. (2012) shows that lamin B2, well known as a nuclear protein, undergoes regulated synthesis in axons, promoting mitochondrial function and axon survival.     

Regulation of local mRNA translation

Regulated local mRNA translation is one mechanism cells employ to concentrate proteins in particular locations. However, cells use many different strategies to accomplish this task; for example, some mRNAs are destroyed in regions where they are not wanted, other mRNAs are repressed in areas where their translation would be deleterious, and yet other mRNAs are transported, in a quiescent state, to the sites where their translation is activated. The importance of local translation cannot be overstated, for, depending on the species or cell type, it is required for cell division, establishment of mating type, development and memory formation.     

Dendritic mRNA: transport, translation and function

Many cellular functions require the synthesis of a specific protein or functional cohort of proteins at a specific time and place in the cell. Local protein synthesis in neuronal dendrites is essential for understanding how neural activity patterns are transduced into persistent changes in synaptic connectivity during cortical development, memory storage and other long-term adaptive brain responses. Regional and temporal changes in protein levels are commonly coordinated by an asymmetric distribution of mRNAs. This Review attempts to integrate current knowledge of dendritic mRNA transport, storage and translation, placing particular emphasis on the coordination of regulation and function during activity-dependent synaptic plasticity in the adult mammalian brain.     

Epsilon as an initiator of translation of CAT mRNA in Escherichia coli

Epsilon sequence (UUAACUUUA) has originally been found in the bacteriophage T7 gene 10 leader region. It enhances translation in Escherichia coli via base pairing with nucleotides 458-466 located in the helical domain #17 of 16S rRNA. We have recently reported that when the complementarity to 16S rRNA is extended, the epsilon is converted from an enhancer to an independent initiator of translation. Here we report the effect of two other structural parameters, positioning in mRNA and the degree of complementarity to 16S rRNA on the translation initiation activity of epsilon in E. coli cells. Our results show that epsilon displays maximal activity as a translational initiator at its natural 9-nucleotide-long complementarity to 16S rRNA and at a 16-nucleotide-long distance to the initiation codon. Under these conditions its efficiency is comparable with that of the consensus Shine-Dalgarno sequence.     

Eucaryotic oligonucleotides affecting mRNA translation

High speed-supernatants and ribosomal salt washes of dormant and developing Artemia salina embryos contain a potent inhibitor of translation; it blocks the elongation factor EF-1-dependent ribosomal binding of aminoacyl-tRNA. A translation activator that counteracts the effect of the inhibitor is found in the same fractions from developing embryos; there is little activator in undeveloped cysts. The appearance of the activator may be responsible for the onset of protein synthesis when development resumes. Both compounds are oligonucleotides. The inhibitor, M_r about 6000, is rich in pyrimidines (47% U, 11% A, 26% C, 16% G), sensitive to RNase A, and resistant to RNase T1. The activator, M_r about 9000, is rich in guanine (33% U, 10% A, 6% C, 51% G), sensitive to RNase T1, and resistant to RNase A. It complexes with the inhibitor and inactivates it. Inhibitor and activator seem to be end products of hydrolysis of embryo RNA by RNase T1 and RNase A, respectively, and ribosomal salt washes of developing embryos have higher RNase A activity than corresponding fractions from dormant cysts.     

Emerging therapeutics targeting mRNA translation

A defining feature of many cancers is deregulated translational control. Typically, this occurs at the level of recruitment of the 40S ribosomes to the 5'-cap of cellular messenger RNAs (mRNAs), the rate-limiting step of protein synthesis, which is controlled by the heterotrimeric eukaryotic initiation complex eIF4F. Thus, eIF4F in particular, and translation initiation in general, represent an exploitable vulnerability and unique opportunity for therapeutic intervention in many transformed cells. In this article, we discuss the development, mode of action and biological activity of a number of small-molecule inhibitors that interrupt PI3K/mTOR signaling control of eIF4F assembly, as well as compounds that more directly block eIF4F activity.     

Reprogramming mRNA translation during stress

The survival of mammalian cells exposed to adverse environmental conditions requires a radical reprogramming of protein translation. Stress-activated kinases target components of the initiation machinery (e.g. eIF2alpha, eIF4E-BP, eIF4B, and ribosomal protein S6) to inhibit the translation of 'housekeeping' proteins and promote the translation of repair enzymes. Accumulating untranslated mRNA is concentrated at stress granules where it is sorted and triaged to sites of storage, reinitiation, or decay. At the same time, the translation of mRNAs encoding repair enzymes is selectively preserved by both internal ribosome entry site-dependent and -independent mechanisms. In combination, these stress-activated processes coordinately reprogram mRNA translation and decay in a way that conserves anabolic energy, preserves essential mRNAs, and promotes the repair of stress-induced molecular damage.     

The role of mRNA competition in regulating translation. III. Comparison of in vitro and in vivo results

Competition of encephalomyocarditis virus, reovirus, and L-cell mRNAs for a message-discriminatory component was studied in vitro. The data were analyzed qualitatively to determine the relative initiation efficiencies among the various mRNAs. The effects of potassium chloride concentration, magnesium acetate concentration, and m7G methylation on mRNA competition in vitro were also studied. These results were correlated with translation rates in vivo for the same mRNAs, to determine if the sites of competition in vitro and in vivo are the same. It was found that under a particular set of magnesium acetate and potassium chloride concentrations, the order of mRNA initiation efficiencies was the same both in vivo and in vitro, suggesting that the same limiting message-discriminatory factor is regulating initiation rates in both cases. This can only be accomplished in a competitive situation when RNA is in molar excess relative to the discriminatory component.