In vitro translation of drosophila heat-shock and non-heat-shock mRNAs in heterologous and homologous cell-free systems

Upon heat shock, Drosophila Kc cells still contain normal cellular messenger RNAs in the cytoplasm. The distribution of these 25°C mRNAs between polysomes and the postpolysomal fraction of heat-shocked cells appears unaltered as compared with control cells. The translatability of these normal cellular messages isolated from heat-shocked and non-heat-shocked Kc cells is unaltered when analyzed by in vitro translation in the rabbit reticulocyte lysate. In contrast, homologous cell-free translation systems obtained from Kc cells effectively discriminate between the in vitro translation of normal cellular messages and heat-shock-specific mRNAs. In particular, a cell-free system from heat-shocked Drosophila Kc cells almost completely shuts down the translation of 25°C messenger RNA species, whereas the translatability of heat-shock-specific messenger RNA appears to be unaffected.     

Cell-free translation systems prepared from starfish oocytes faithfully reflect in vivo activity; mRNA and initiation factors stimulate supernatants from immature oocytes.

Meiotic maturation stimulates a change in the translation of stored mRNAs: mRNAs encoding proteins needed for growth of oocytes are translated before meiotic maturation, whereas those encoding proteins required for cleavage are translated after meiotic maturation. Studies of translational regulation during meiotic maturation have been limited by the lack of translationally active cell-free supernatants. Starfish oocytes are ideal for preparing cell-free translation systems because experimental application of the hormone 1-methyladenine induces their maturation, synchronizing meiosis. We have prepared such systems from both immature and mature oocytes of starfish. Changes in protein synthesis rates and the specificity of proteins synthesized in these cell-free translation supernatants mimic those seen in vivo. Supernatants both from immature and mature oocytes have a high capacity to initiate new translation because 90% of the proteins made are newly initiated from mRNAs. Cell-free supernatants from mature oocytes have a much higher rate of initiation of translation than those from immature oocytes and use the 43S preinitiation complexes more efficiently in initiation of translation. Similarly, we have shown that mRNAs and initiation factors are rate limiting in cell-free translation systems prepared from immature oocytes. In addition, cell-free translation systems prepared from immature oocytes are only slightly, if at all, inhibitory to cell-free translation systems from mature oocytes. Thus, soluble inhibitors, if they exist, are rapidly converted by cell-free supernatants from mature oocytes. The similarities between translation in our starfish cell-free translation systems and in intact oocytes suggests that the cell-free translation systems will be useful tools for further studies of maturation events and translational control during meiosis.     

Regulation of protein synthesis in heat-shocked Drosophila cells. Soluble factors control translation in vitro

We have developed an in vitro translation system from heat-shocked and normal Drosophila cultured cells. The lysates retain regulation of translation typical of the whole cells from which they were prepared, both when programmed by endogenous mRNA and when RNA-dependent. These systems have been used to investigate the mechanism of shutdown of normal protein synthesis and selection of heat shock mRNAs for translation in heat shock in Drosophila. Supplementation of intact RNA-dependent lysates with separated ribosome or supernatant fractions from normal or heat-shocked translation systems showed the normal supernatant fraction could "rescue" normal protein synthesis in a heat shock lysate. Normal ribosomes had no rescuing activity and neither heat shock fraction affected translation in normal lysates. Reconstitution of the system from separated ribosomes and supernatant in normal and mixed combinations showed heat shock and normal ribosomes were both competent to support normal protein synthesis with normal supernatant. Heat shock supernatant did not support normal protein synthesis with ribosomes from either source. We conclude that the factors regulating translation in heat-shocked Drosophila cells are soluble factors in the lysate and that the soluble factors present in the normal lysate are dominant.     

Translational control in oocyte development

Translational control of specific mRNAs is a widespread mechanism of gene regulation, and it is especially important in pattern formation in the oocytes of organisms in which the embryonic axes are established maternally. Drosophila and Xenopus have been especially valuable in elucidating the relevant molecular mechanisms. Here, we comprehensively review what is known about translational control in these two systems, focusing on examples that illustrate key concepts that have emerged. We focus on protein-mediated translational control, rather than regulation mediated by small RNAs, as the former appears to be predominant in controlling these developmental events. Mechanisms that modulate the ability of the specific mRNAs to be recruited to the ribosome, that regulate polyadenylation of specific mRNAs, or that control the association of particular mRNAs into translationally inert ribonucleoprotein complexes will all be discussed.     

Mechanisms of translational regulation in Drosophila

Translational regulation plays an essential role in many phases of the Drosophila life cycle. During embryogenesis, specification of the developing body pattern requires co-ordination of the translation of oskar, gurken and nanos mRNAs with their subcellular localization. In addition, dosage compensation is controlled by Sex-lethal-mediated translational regulation while dFMR1 (the Drosophila homologue of the fragile X mental retardation protein) controls translation of various mRNAs which function in the nervous system. Here we describe some of the mechanisms that are utilized to regulate these various processes. Our review highlights the complexity that can be involved with multiple factors employing different mechanisms to control the translation of a single mRNA.     

Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs

The ability to map the position of ribosomes and their associated factors on mRNAs is critical for an understanding of translation mechanisms. Earlier approaches to monitoring these important cellular events characterized nucleotide sequences rendered nuclease-resistant by ribosome binding. While these approaches furthered our understanding of translation initiation and ribosome pausing, the pertinent techniques were technically challenging and not widely applied. Here we describe an alternative assay for determining the mRNA sites at which ribosomes or other factors are bound. This approach uses primer extension inhibition, or "toeprinting," to map the 3' boundaries of mRNA-associated complexes. This methodology, previously used to characterize initiation mechanisms in prokaryotic and eukaryotic systems, is used here to gain an understanding of two interesting translational regulatory phenomena in the fungi Neurospora crassa and Saccharomyces cerevisiae: (a) regulation of translation in response to arginine concentration by an evolutionarily conserved upstream open reading frame, and (b) atypical termination events that occur as a consequence of the presence of premature stop codons.     

Translational control of development inC. elegans

Translational control by the 3′untranslated regions (3′UTRs) of mRNAs contributes to important events throughout the development of C. elegans. In oocytes and early embryos, maternal mRNAs are controlled by 3′UTR elements to restrict translation of their protein products to specific blastomeres. Localized translation is probably critical for specifying blastomere identity. In both germline and somatic cells, mRNAs from sex determining genes are translationally repressed by 3′UTR controls. These controls balance the activities that specify male and female cell fates. During larval development, the temporal sequence of cell lineages requires 3′UTR-mediated regulation of heterochronic genes by a small non-protein coding RNA. We review what is known about these translational control mechanisms in C. elegans. This overview illustrates that translational control by 3′UTR elements is a powerful mechanism for regulating the expression of multiple gene products in diverse cell types during development of a multi-cellular animal.     

Relative importance of 7-methylguanosine in ribosome binding and translation of vesicular stomatitis virus mRNA in wheat germ and reticulocyte cell-free systems.

Vesicular stomatitis virus mRNAs with these four types of 5'-termini, (a) m7G5'ppp5'(m)Am, (b) ppp5'(m)Am, (c) m7G5'-ppp5' Am, and (d) G5'ppp5'A, were prepared and their translation and ribosome binding analyzed in wheat germ and reticulocyte cell-free protein synthesis systems. The relative efficiencies of translation of individual vesicular stomatitis virus (VSV) mRNAs having type 2 termini ranged from 23 to 29% of the control (type 1) RNA in the reticulocyte system and 6 to 7% of control RNA in the wheat germ system. A similar difference between the two systems was seen in ribosome-binding experiments in which type 2 RNA formed an 80 S initiation complex with high efficiency (70% of control type 1 RNA) in the reticulocyte system, but with low efficiency (17% of control RNA) in the wheat germ system. Similar differences in the importance of m7G in translation in the two systems were seen when VSV mRNAs synthesized in vitro with type 3 and type 4 termini were analyzed. However, the analysis of type 4 RNA (which was synthesized in vitro in the presence of S-adenosylhomocysteine) was complicated by the presence of abnormally large poly(A) at its 3'-end. Another series of experiments showed that compounds such as 5'pm7G and m7G5'ppp5'Np are potent and specific inhibitors of translation of all types of VSV mRNAs in the wheat germ system (greater than 98% inhibition) but cause less than 20% inhibition of translation in the reticulocyte system. Taken together, all of the results indicate that a 5'-terminal m7G is far more important in translation of VSV mRNAs in the heterologous plant cell-free system than in the reticulocyte lysate system.     

Proteomic analysis of reaper 5' untranslated region-interacting factors isolated by tobramycin affinity-selection reveals a role for La antigen in reaper mRNA translation

Translational control is a key step in gene expression regulation during apoptosis. To understand the mechanisms of mRNA translation of a pro-apoptotic gene, reaper (rpr), we adapted the tobramycin-aptamer technique described by Hartmuth et al. (Proc. Natl. Acad. Sci. USA 2002, 99, 16719-16724) for the analysis of proteins interacting with rpr 5' untranslated region (UTR). We assembled ribonucleoprotein complexes in vitro using translation extracts derived from Drosophila embryos and purified the RNA-protein complexes for mas spectrometry analysis. We identified the proteins bound to the 5' UTR of rpr. One of them, the La antigen, was validated by RNA-crosslinking experiments using recombinant protein and by the translation efficiency of reporter mRNAs in Drosophila cells after RNAinterference experiments. Our data provide evidence of the involvement of La antigen in the translation of rpr and set a protocol for purification of tagged-RNA-protein complexes from cytoplasmic extracts.     

Translational control of growth factor and proto-oncogene expression

Control of translation is now understood to be one of the major regulatory events in eukaryotic gene expression. Moreover there is evidence which suggests that aberrant expression of growth-related genes by translational mechanisms makes a significant contribution to cell transformation. However, the mechanisms which regulate translation of specific growth-related mRNAs have yet to be fully elucidated. The majority of these mRNAs have long 5' untranslated regions (UTRs) and three features which are important in translational control have been identified, namely (i) structured regions which inhibit the scanning mechanisms of translation, (ii) regulatory upstream open reading frames and (iii) internal ribosome entry segments which are capable of initiating cap-independent translation. In this review the translational regulation of specific mRNAs encoding growth factors and proto-oncogenes by these three mechanisms will be discussed, together with examples of altered translational regulation in neoplasia.     

Diversity in translational regulation

Translational control of individual mRNAs relies on cis-regulatory elements, which are often found in the 3' untranslated region. The best characterized of these regulate cytoplasmic polyadenylation, and much of this process can now be defined in terms of molecular interactions, protein modifications and their consequences. Biochemical and genetic approaches have advanced the understanding of the many instances of translational regulation that are crucial for body patterning in Drosophila. For example, in vitro translation systems have been used to study the regulatory mechanisms, and genetic interactions have been instrumental in establishing a link between a regulatory factor and a component of the translational apparatus. Although most examples of control are thought to affect the initiation of translation, two classes of regulatory factors, one a protein and one a short non-coding RNA now appear to inhibit protein synthesis during elongation. Diversity seems to be a central feature of translational control, both in the mechanisms themselves and in the situations where this form of regulation is used.     

Signalling to translation: how signal transduction pathways control the protein synthetic machinery

Recent advances in our understanding of both the regulation of components of the translational machinery and the upstream signalling pathways that modulate them have provided important new insights into the mechanisms by which hormones, growth factors, nutrients and cellular energy status control protein synthesis in mammalian cells. The importance of proper control of mRNA translation is strikingly illustrated by the fact that defects in this process or its control are implicated in a number of disease states, such as cancer, tissue hypertrophy and neurodegeneration. Signalling pathways such as those involving mTOR (mammalian target of rapamycin) and mitogen-activated protein kinases modulate the phosphorylation of translation factors, the activities of the protein kinases that act upon them and the association of RNA-binding proteins with specific mRNAs. These effects contribute both to the overall control of protein synthesis (which is linked to cell growth) and to the modulation of the translation or stability of specific mRNAs. However, important questions remain about both the contributions of individual regulatory events to the control of general protein synthesis and the mechanisms by which the translation of specific mRNAs is controlled.