Translation Of Exogenous mRNA Research Articles

Quaternization drives spleen-to-lung tropism conversion for mRNA-loaded lipid-like nanoassemblies.

Background: As the overwhelming majority of advanced mRNA delivery systems are preferentially accumulated in the liver, there is an accelerating growth in the demand for the development of non-liver mRNA delivery platforms. Methods: In this study, we prepared cationic lipid-like nanoassemblies through a N-quaternizing strategy. Their physicochemical properties, in vitro mRNA delivery efficiency, and organ tropism in mice were investigated. Results: Introduction of quaternary ammonium groups onto lipid-like nanoassemblies not only enhances their mRNA delivery performance in vitro, but also completely alters their tropism from the spleen to the lung after intravenous administration in mice. Quaternized lipid-like nanoassemblies exhibit ultra-high specificity to the lung and are predominantly taken up by pulmonary immune cells, leading to over 95% of exogenous mRNA translation in the lungs. Such mRNA delivery carriers are stable even after more than one-year storage at ambient temperature. Conclusions: Quaternization provides an alternative method for design of new lung-targeted mRNA delivery systems without incorporation of targeting ligands, which should extend the therapeutic applicability of mRNA to lung diseases.

Exploiting exercise and mTOR enhanced protein synthesis to augment mRNA vaccine translation

Exercise has been previously proposed as an adjuvant for traditional vaccines through mechanisms of lymph movement and skeletal muscle damage. However, the novel mRNA technology being used in COVID‐19 and other novel vaccines fundamentally changes how we should consider muscle’s active role in protein production and subsequent protective immunity. Following exercise, mammalian target of rapamycin (mTOR) activation drives total protein translation increases in skeletal muscle. We hypothesize that this 4‐24 hour window of opportunity can be exploited to augment exogenous mRNA translation. To evaluate the feasibility of this approach, we first interrogated mTOR using fully differentiated C2C12 mouse skeletal muscle myotubes that were transfected with mRNA encoding for enhanced green fluorescent protein (eGFP) or FLAG‐tagged Comirnaty SARS‐CoV‐2 Spike sequence in mTOR modulating conditions. Myotube transfection in the presence of rapamycin (Figure 1A) and resveratrol (Figure 1B) showed significant decreases in protein translation as measured by eGFP fluorescence, indicating that exogenous mRNA translation can be decreased pharmacologically by inhibiting the mTOR pathway. To simulate exercise enhanced protein synthesis, we treated myotubes with insulin‐like growth‐factor 1 (IGF‐1), as this protein is not only upregulated following exercise but also signals through an overlapping pathway with mechanoreceptors. Transfections with mRNA encoding for either eGFP or Comirnaty‐FLAG during IGF‐1 treatment showed a dose dependent increase in eGFP fluorescence (Figure 1C) and a significant increase in Comirnaty‐FLAG translation as measured by ELISA (Figure 1D). Current studies are investigating the use of electrical stimulation induced contraction on C2C12 myotubes prior to transfection as well as in vivo models of exercise and intramuscular mRNA injections. The implications of these results may impact future mRNA therapies to utilize exercise, or modulation of intracellular signaling pathways affected by exercise, as a method to improve vaccine‐induced exogenous protein production.

Expression of exogenous fluorescent proteins in early freshwater pond snail embryos

We have for the first time succeeded in expressing in vitro-synthesized mRNAs in both the sinistral and the dextral Lymnaea stagnalis early embryos by microinjecting the mRNAs into the eggs before the first polar body stage. Translation of exogenous mRNA in developing embryos was confirmed by expressing various fluorescent proteins; mCherry, DsRed-Express, and enhanced green fluorescent protein. We have found that the protein expression derived from the introduced exogenous mRNA largely depends on the elapsed time after the microinjection and not on the developmental stage of injection, and also on the amount of injected mRNA. Developmental abnormalities were hardly observed. The first notable fluorescent signal was detected within 2-3 h after the injection while the embryos were still in uncleaved stage. Fluorescence gradually increased until 8-9 h and was stable up to 24 h. From these results, it is suggested that there is enough translation machinery necessary for early development and the translation of injected mRNA proceeds immediately and constantly in the early embryos. This is true for both the sinistral and dextral L. stagnalis embryos. Application of the developed method to other freshwater pond snails, dextral Lymnaea peregra, sinistral Physa acuta, and sinistral Indoplanorbis exustus revealed that their early expression mechanisms to be similar to that of L. stagnalis. Thus, in vitro-synthesized mRNA expression is expected to be important for the understanding of evolutional process and the molecular mechanism underlining the handedness determination in these freshwater snail embryos.

Viral Translation Is Coupled to Transcription in Sindbis Virus-Infected Cells

During the late phase of Sindbis virus infection, the viral subgenomic mRNA is translated efficiently in BHK cells, whereas host protein synthesis is inhibited. However, transfection of in vitro-generated Sindbis virus subgenomic mRNA leads to efficient translation in uninfected BHK cells, whereas it is a poor substrate in infected cells. Therefore, the structure of the subgenomic mRNA itself is not sufficient to confer its translatability in infected cells. In this regard, translation of the subgenomic mRNA requires synthesis from the viral transcription machinery. The lack of translation of transfected viral mRNAs in infected cells is not due to their degradation nor is it a consequence of competition between viral transcripts and transfected mRNAs, because a replicon that cannot produce subgenomic mRNA also interferes with exogenous mRNA translation. Interestingly, subgenomic mRNA is translated more efficiently when it is transfected into uninfected cells than when it is transcribed from a transfected replicon. Finally, a similar behavior was observed for other RNA viruses, such as vesicular stomatitis virus and encephalomyocarditis virus. These findings support the notion that translation is coupled to transcription in cells infected with different animal viruses.

Further evidence that ribavirin interacts with eIF4E

This commentary discusses the recent reports in RNA by Yan and colleagues and Westman and colleagues of the apparent failure of ribavirin to bind to recombinant eIF4E and inhibit 7-methyl guanosine cap-dependent exogenous mRNA translation of cell extracts in vitro. Measuring binding by using affinity chromatography of matrix-immobilized proteins and by using protein emission fluorescence spectroscopy in the presence of nucleotide ligands, as well as limitations of using cell extracts for the assessment of mechanisms of mRNA translation are discussed. Possible reasons for the discordant findings of Yan and colleagues and Westman and colleagues are suggested, and direct observation of the specific binding of ribavirin to eIF4E by using mass spectrometry is presented.

Xenopus NF-Y pre-sets chromatin to potentiate p300 and acetylation-responsive transcription from the Xenopus hsp70 promoter in vivo.

We identify Xenopus NF-Y as a key regulator of acetylation responsiveness for the Xenopus hsp70 promoter within chromatin assembled in Xenopus oocyte nuclei. Y-box sequences are required for the assembly of DNase I-hypersensitive sites in the hsp70 promoter, and for transcriptional activation both by inhibitors of histone deacetylase and by the p300 acetyltransferase. The viral oncoprotein E1A interferes with both of these activation steps. We clone Xenopus NF-YA, NF-YB and NF-YC and establish that NF-Y is the predominant Y-box-binding protein in Xenopus oocyte nuclei. NF-Y interacts with p300 in vivo and is itself a target for acetylation by p300. Transcription from the hsp70 promoter in chromatin can be enhanced further by heat shock factor. We suggest two steps in chromatin modification at the Xenopus hsp70 promoter: first the binding of NF-Y to the Y-boxes to pre-set chromatin and second the recruitment of p300 to modulate transcriptional activity.

Preparation of a Cell-Free Translation System with Minimal Loss of Initiation Factor eIF-2/eIF-2B Activity

Cell-free translation systems prepared from suspension-cultured HeLa S3 cells or mouse L cells by hypotonic shock followed by Dounce homogenization poorly initiated the translation of exogenous mRNA. In contrast, cell extracts prepared from cells exposed to the detergent lysolecithin translated exogenous mRNA readily. The block in initiation in the former lysates was localized to the ribosome fraction. During in vitro translation polysomes from homogenized cells disaggregated but the run-off ribosomes were unable to reinitiate translation. The block resulted from a decrease in eukaryotic initiation factor 2 (eIF-2) or the guanine nucleotide exchange factor (eIF-213) activity, since the addition of eIF-2 or eIF-2B to these latter extracts substantially improved the capacity of the extract to initiate translation of exogenous mRNA. Extracts from homogenized cells, but not from detergent-treated cells, showed enhanced ability to phosphorylate the α subunit of exogenous eIF-2. We show that the method of cell extract preparation greatly influences the state of eIF2/eIF-2B activity in the resulting extract and that extracts in which this activity is maintained can readily initiate translation on exogenous mRNA and reinitiate on endogenous mRNA.

MRNA-directed synthesis and insertion of functional amino acid receptors in Xenopus laevis oocytes.

A major goal for neuroscientists over the past 1&20 years has been the understanding of the mechanism and control of membrane excitability in the mammalian central nervous system (CNS). To realize this ambition, various methods and techniques have been evolved to study drugand voltage-operated membrane ion channels, which have required the manipulation of CNS tissue. A large majority of these techniques, both biochemical and electrophysiological, have involved the use of brain slices, membrane homogenates and also dissociated and organotypic tissue culture preparations. In all three situations, the drug receptors and associated ionophores resided in the native neuronal/glial membrane and were not always ideally placed for study. As an alternative, we have effectively transferred some of the apparatus mediating CNS membrane excitability, such as certain drug receptors and voltage-activated ion channels, from their neuronal environment, into the cell membrane of the Xenopus oocyte, by utilizing the translation of exogenous mRNA. This preparation has been a highly convenient medium for electrophysiological and radioligand-binding studies on drug

The translational inhibitor 10 S cytoplasmic ribonucleoprotein of chick embryonic muscle. Dissociation and reassociation.

Cytoplasmic 10S ribonucleoprotein (iRNP) is a potent inhibitor of mRNA translation in vitro and contains a 4 S translation inhibitory RNA species (iRNA) (Sarkar, S., Mukherjee, A. K., and Guha, C. (1981) J. Biol. Chem. 256, 5077-5086). This ribonucleoprotein has now been resolved into protein and RNA components by DEAE-cellulose chromatography in the absence of both K+ and Mg2+ ions. These cations are required for maintaining the nucleoprotein structure of iRNP. Incubation of the dissociated protein and RNA components in the presence of K+ and Mg2+ at 35 degrees C reconstitutes a 10 S particle which is indistinguishable from native iRNP with respect to the elution profile by gel filtration, UV spectra, buoyant density, resistance to pancreatic RNase, and ability to inhibit exogenous mRNA translation in vitro. Chick muscle tRNA and globin mRNA could not form an RNP complex with the protein moieties of iRNP. The separated proteins, unlike iRNA and iRNP, do not inhibit mRNA translation. Their function may be to protect iRNA from ribonuclease digestion, since iRNP is ribonuclease-resistant. The ability to dissociate the iRNP particle and to specifically reconstitute it from the separated components indicates that it is a unique cellular entity which is distinct from other ribonucleoproteins.

The effect of elevated temperature on protein synthesis in cell-free extracts of cultured Chinese hamster ovary cells

The effect of elevated temperature on the activity of various components involved in protein synthesis was investigated in extracts from cultured Chinese hamster ovary cells. The translation of exogenous mRNA was markedly inhibited by preincubation of the extract for 15 to 20 minutes at 42°C. However, the following intermediary reactions were not affected, or only slightly inhibited, at 42°C: 1) the incorporation of Met-tRNA f into eIF-2·Met-tRNA f·GTP ternary complex; 2) the interaction of the ternary complex with 40S ribosomal subunits to form the 40S preinitiation intermediate; 3) the binding of mRNA and 60S subunits to form the 80S initiation complex; and 4) the reactions catalyzed by elongation factors EF-1 and EF-2. The activity of Met-tRNA synthetase was markedly inhibited, affecting the formation of initiator Met-tRNA f required for the initiation of protein synthesis and the translation of natural mRNA. Other aminoacyl-tRNA synthetases were not significantly affected by the elevated temperature.

Cell-free translation of exogenous mRNA in extracts from dry pea primary axes

Extracts from the primary axes of dry pea (Pisum sativum L.) seeds are able to perform an initiation-dependent translation of exogenous mRNA. SDS polyacrylamide gel electrophoresis of the products synthesized under direction of alfalfa mosaic virus RNA (AMV-RNA) and tobacco mosaic virus RNA (TMV-RNA) shows that the fidelity of translation in this pea system is at least as high as in a wheat embryo cell-free protein synthesizing system. The endogenous messengers are also efficiently translated in extracts from the primary axes of pea seeds. The direct translation of these messengers in a homologous cell-free system may be of interest for a study of the products coded for by the long-lived messengers present in this plant.

Inhibition of exogenous mRNA translation in a cell-free system by chromatin peptides from calf thymus.

Low molecular weight chromatin peptides isolated from calf thymus by affinity chromatography on DNA-cellulose inhibit significantly translation of exogenous isolated mRNA in a reticulocyte cell-free system. Translation with endogenous mRNA present in the system is not inhibited by low peptide concentrations. The data obtained combined with the previous findings suggest that chromatin peptides control gene expression at two levels: transcription and translation.

Rate-limiting factors for lymphocyte protein synthesis Ribosome commitment and the capacity of lymphocyte cell-free systems to translate exogenous mRNAs

The properties of the protein synthesising systems of different lymphocyte preparations have been compared with those of non-lymphoid tissues. Polysome profiles from rat thymocytes, sheep mesenteric lymphocytes, rat liver and mouse ascites tumours showed that the commitment of ribosomes to protein synthesis in lymphocytes was relatively low. Initiation factor activities, assessed on the abilities of post-mitochondrial fractions to support exogenous mRNA translation, were limited or undetectable in lymphoid tissues. While the thymocyte system translated globin mRNA, the response was enhanced by ascites extracts rich in initiation factors. The mesenteric lymphocyte system responded only marginally to globin mRNA and poly(U) but the responses were not enhanced by ascites extracts. The activity of isolated mesenteric ribosomes was comparable with ribosomes from other tissues, indicating that extraribosomal factors were responsible for the poor overall activity of the mesenteric system. Finally, the effects of cycloheximide on the recruitment of polysomes in lymphocytes indicated that the commitment of ribosomes to protein synthesis might be restricted by both limited mRNA availability and limited capacity for initiation of mRNA translation.

Translation of exogenous mRNA in a wheat embryo cell-free system; Effect of composition of cell-free system

Translation of exogenous mRNA in a wheat embryo cell-free system; Effect of composition of cell-free system

Fucose incorporation into oocyte-synthesized rat immunoglobulins

The translation of exogenous mRNA in Xenopus laevis oocytes is accompanied by extensive posttranslational modification of synthesised polypeptides [l] . Synthesis of immunoglobulin heavy and light chains in microinjected oocytes is followed by their assembly into 7 S H& molecules [2] such that specific antibody activity can be demonstrated [31* In order to study further the post-translational modifications of oocyte-syntbesised polypeptides the incorporation and distribution of L-fucose into immunoglobulins was determined.

Release of Interferon-Induced Translation Inhibition by tRNA in Cell-Free Extracts from Mouse Erythroleukemia Cells

Preincubated cell-free lysates from interferon-treated Friend-virus-transformed mouse proerythro-blasts have been used to study the mechanism of the antiviral effect of mouse interferon. Similar to preincubated cell-free protein-synthesizing systems derived from other interferon-treated murine cells, translation of globin and encephalomyocarditis virus (EMC) RNA is inhibited. In a fractionated cell-free system run-off ribosomes prepared from interferon-treated and control proerythroblasts were of the same activity. Addition of tRNA from eukaryotic cells to the reaction mixture could restore the translation capacity for EMC and globin RNA. Various purified tRNA species were tested for their capacity to restore the translation activity: tRNALys2 from rabbit liver but not the isoaccepting tRNALys3 showed activity to release the inhibition. The efficiency to restore the translation activity was, however, less than with total tRNA from rabbit liver, indicating that other tRNA species are also able to restore the translation activity in extracts from interferon-treated mouse proerythroblasts. tRNAVal1 and partially purified tRNASer and tRNALeu species from rabbit liver had no restoring activity. tRNA modified by periodate treatment could also restore translation activity, although with reduced efficiency. The translation inhibition and release by tRNA in preincubated extracts from interferon-treated mouse proerythroblasts and uninfected L-cells seems to be caused basically by the same molecular mechanism. This phenomenon, however, clearly contrasts with the inhibition of translation of exogenous mRNA observed with extracts from interferon-treated and vaccinia-infected L-cells. The translation inhibition in extracts from interferon-treated and vaccinia-infected cells cannot be reversed by tRNA and an alternative molecular mechanism must therefore be considered.

Interferon Treatment of Ehrlich Ascites Tumor Cells: Effects on Exogenous mRNA Translation and tRNA Inactivation in the Cell Extract

We reported earlier that in cell extracts that were prepared from interferon-treated Ehrlich ascites tumor cells and preincubated and passed through Sephadex G-25 (S60INT), the translation of exogenous mRNA (viral and host) was impaired and the impairment could be overcome to a large extent by adding a crude tRNA preparation from Ehrlich ascites tumor cells but not from Escherichia coli. We find now that the rate of inactivation of some tRNA's (especially those specific for leucine, lysine, and serine) but not those of many others is faster in S30INT than in corresponding extracts from control cells. This increased rate of tRNA inactivation may perhaps account for the need for added RNA to overcome at least partially the impairment of translation in S30INT. The relationship of the increased rate of tRNA inactivation to the antiviral effect of interferon is unclear. So far no significant difference has been detected in the amount of tRNA needed to overcome the impairment of encephalomyocarditis virus RNA translation in S30INT between tRNA from interferon-treated cells and tRNA from control cells. Futhermore, no difference was found in the rate of inactivation in S30INT between leucine-specific tRNA's from interferon-treated and from control cells. tRNA's specific for leucine and lysine were not inactivated (unless very slowly) during incubation under out conditions in an extract from interferon-treated (or from control) cells unless the extract had been passed through Sephadex G-25 or dialyzed. The translation fo exogenous mRNA was, however, impaired in an extract from interferon-treated cells that had not been passed through Sephadex G-25. This impairment was apparently not overcome by added tRNA.

Synthesis of plant virus coat proteins in an animal cell-free system.

CELL-FREE systems which are capable of accurate translation of exogenous mRNA have proved invaluable tools for studying the mechanism of protein synthesis both in prokaryotes1 and, more recently, in eukaryotes2. Since the availability of suitable mRNAs is often a limiting factor in such studies, RNA-containing viruses have been widely used as sources of RNA for translation in bacterial, plant and animal cell-free systems; for example, the bacteriophages f2 (ref. 3) and Qβ (ref. 4), the plant viruses brome mosaic virus5 and satellite tobacco necrosis virus6 and the picornaviruses, encephalomyocarditis virus7 and mengo virus8. But, although picornavirus RNA is actively translated in animal cell-free systems, it is not an ideal messenger since its large size9 and single polypeptide chain initiation sit10, 11 hinder complete translation in vitro12. As alternative sources of mRNA for animal cell-free systems, we have studied the plant viruses brome mosaic virus (BMV) and tobacco rattle virus (TRV). Here we report the efficient and accurate synthesis of the coat proteins of these viruses in a mammalian cell-free system directed by the viral RNAs.

Increased efficiency of exogenous messenger RNA translation in a Krebs ascites cell lysate.

Addition of a 0.5 M KCl wash fraction from rabbit reticulocyte ribosomes causes a 3- to 10-fold increase in the extent of translation of natural mRNAs by Krebs-cell lysates. In the presence of the wash fraction, 1 pmol of rabbit or mouse 10S RNA directs the incorporation of 80 pmol of leucine into rabbit globin. The addition of human 10S RNA results in the synthesis of equal amounts of human alpha and beta chains, identified by column chromatography. The stimulation by the wash fraction is almost completely dependent on added mammalian tRNA. In contrast to the wash fraction from rabbit reticulocytes, the wash fraction isolated from Krebs-cell ribosomes is inhibitory to both endogenous and exogenous mRNA translation. The stimulation by the wash fraction from rabbit ribosomes is not specific for globin mRNAs, but also increases endogenous, phage Qbeta, and viral RNA-directed protein synthesis.

Translation of exogenous messenger RNA for hemoglobin on reticulocyte and liver ribosomes.

The ribosome and initiation factor requirements for translation of rabbit-reticulocyte hemoglobin mRNA on rabbit reticulocyte ribosomes, reticulocyte ribosomal subunits, and liver ribosomes have been studied. Excellent synthesis of globin chains from exogenous mRNA in the fractionated cell-free system has been achieved. There is a near absolute requirement for each of the initiation factors, M(1), M(2), and M(3) (as well as for the supernatant proteins) for the translation of exogenous mRNA. Liver microsomal wash will partially replace reticulocyte factors M(1) and M(2), but will not replace the requirement for reticulocyte factor M(3). Rabbit liver ribosomes and rabbit reticulocyte ribosomes are equally active in their ability to support the translation of exogenous hemoglobin mRNA.