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PubMed2025

Translation of the downstream ORF from bicistronic mRNAs by human cells: Impact of codon usage and splicing in the upstream ORF.

Biochemistry textbooks describe eukaryotic mRNAs as monocistronic. However, increasing evidence reveals the widespread presence and translation of upstream open reading frames preceding the "main" ORF. DNA and RNA viruses infecting eukaryotes often produce polycistronic mRNAs and viruses have evolved multiple ways of manipulating the host's translation machinery. Here, we introduce an experimental model to study gene expression regulation from virus-like bicistronic mRNAs in human cells. The model consists of a short upstream ORF and a reporter downstream ORF encoding a fluorescent protein. We have engineered synonymous variants of the upstream ORF to explore large parameter space, including codon usage preferences, mRNA folding features, and splicing propensity. We show that human translation machinery can translate the downstream ORF from bicistronic mRNAs, albeit reporter protein levels are thousand times lower than those from the upstream ORF. Furthermore, synonymous recoding of the upstream ORF exclusively during elongation significantly influences its own translation efficiency, reveals cryptic splice signals, and modulates the probability of downstream ORF translation. Our results are consistent with a leaky scanning mechanism facilitating downstream ORF translation from bicistronic mRNAs in human cells, offering new insights into the role of upstream ORFs in translation regulation.

Humans↗

PubMed1977

Somatic nuclei in amphibian oocytes: evidence for selective gene expression.

Previous work has shown that multiple HeLa nuclei injected into Xenopus oocytes remain transcriptionally active for many days and that the expression of HeLa genes in oocytes can be detected by 2-D gel electrophoresis. We show here that of 25 proteins which have the electrophoretic properties of HeLa gene products, only 3 are expressed in injected oocytes. To test that these proteins are products of HeLa genes, and not products of activated oocyte genes, we have injected HeLa nuclei into enucleated oocytes. Three days later, several HeLa proteins were synthesized. The turning off of most HeLa genes in injected oocytes is apparently not at the translational level. This is indicated by the fact that adenovirus mRNA is efficiently translated when injected into Xenopus oocytes. When adenovirus-infected HeLa cells nuclei are injected into oocytes the adenovirus genes are not expressed, although some HeLa genes are expressed by the same nuclei. The same HeLa genes as are expressed or switched off in injected Xenopus oocytes are also preferentially expressed or switched off in injected oocytes of a Urodele amphibian, Pleurodeles. This suggests that conditions or molecules may exist in oocytes which selectively impose on injected nuclei a new programme of gene expression.

Adenoviridae↗

PubMedDate not supplied

In vitro translation of natural mRNAs in a cell-free system containing components from interferon-treated chicken fibroblasts and factor preparations from mouse ascites cells or rabbit reticulocytes.

The effect of interferon has been studied in a mixed cell-free protein synthesizing system. Hemoglobin (Hb) and Encephalomyocarditis virus (EMC)-RNA can be efficiently translated in vitro in a system containing S-30 lysates or run-off ribosomes from primary chick embryo fibroblasts (CEF) and a postmicrosomal supernatant from mouse ascites cells or a ribosomal-wash preparation from rabbit reticulocytes. Ribosomes prepared from CEF pretreated with high doses of homologous interferon (500 units/ml) were able to translate Hb-RNA in the presence of heterologous factors with the same efficiency as ribosomes prepared from control cells. Translation of EMC-RNAP WAS SLIGHTLY Reduced if ribosomes from interferon-treated cells were used in the mixed cell-free system, confirming previous reports. No inhibitory effect caused by interferon treatment of CEF cells could be detected on in vitro translation of natural mRNAs if the cells had, in addition to interferon treatment, been infected with vaccinia virus. Possible reasons for the different observations made with out cell-free protein synthesizing system from CEF and with cell-free systems prepared from mouse cells are discussed.

Animals↗

PubMed2026

Involvement of tRNA thiolation in uORF-mediated translational regulation during Xylogenesis in Arabidopsis thaliana.

Post-transcriptional modification of tRNAs is an important mechanism for regulating translation efficiency and cellular homeostasis, yet its contribution to upstream open reading frame (uORF)-mediated translational control remains largely unexplored. In this study, we investigated the role of tRNA thiolation in thermospermine-dependent regulation of xylem development in Arabidopsis thaliana. Using a suppressor screen of the thermospermine-deficient mutant acaulis5 (acl5), which exhibits dwarfism and excessive xylem differentiation, we identified suppressor-of-acl502 (sac502) as a recessive loss-of-function allele of CTU2, a gene encoding a key enzyme in the biosynthesis of the wobble uridine modification 5-methoxycarbonylmethyl-2-thiouridine. Mutations in other components of the same modification pathway, including ROL5 and TRM9, similarly suppressed the acl5 phenotype. Translational analyses using 5' leader-GUS reporter constructs revealed that the ctu2 mutation did not enhance translation of the mRNA containing a thermospermine-responsive uORF of SAC51, but instead significantly reduced translation of that of SACL3, a member of the SAC51 family, and that of LONESOME HIGHWAY (LHW), which contains another conserved uORF in the 5' leader region. Polysome profiling further demonstrated decreased association of SACL3 and LHW mRNAs with actively translating ribosomes in ctu2. Genetic interaction analyses supported the conclusion that the suppression of excessive xylem formation in acl5 by ctu2 is attributable to reduced LHW activity. In addition, ctu2 mutants displayed increased sensitivity to exogenous thermospermine, resembling the response of lhw mutants. Together, our results reveal that tRNA thiolation contributes to uORF-mediated translational regulation of key developmental regulators and identify tRNA modification as an important regulatory layer controlling vascular development.

Arabidopsis↗

PubMed1977

Altered surface properties of Escherichia coli associated with a specific amino acid change in the S12 ribosomal protein of streptomycin-resistant mutants.

Escherichia coli mutants resistant to streptomycin exhibited differences in countercurrent distribution from the parental strains. The degree of difference from the parental strain correlated with the degree of restriction of translation and thus the particular strA allele. The changes in countercurrent distribution in the phase systems used probably resulted predominantly from surface charge alterations. The differences in countercurrent distribution in these and other mutants may be a useful selective technique to obtain different types of mutants for which specific selective techniques may not be available. In addition, it appears that the surface properties of cells, which determine their position in countercurrent distribution, are a function of the translational efficiency and fidelity, and that the surface of cells consists of a mosaic that is an expression of this translational fidelity.

Countercurrent Distribution↗

PubMed2025

Unconventional codon usage bias mediates mRNA translational dynamics in macrophages.

Macrophages require rapid and tightly controlled regulatory mechanisms to respond to environmental disruptions. While transcriptional regulation has been well characterized, the mechanisms underlying translational control in macrophages remain poorly understood. Here, we investigated the dynamics of mRNA translation in mouse macrophages during acute, intermediate, and prolonged LPS exposure. Our results reveal clear phase-specific translational regulation during macrophage polarization, which initially increases the synthesis of inflammatory mediators and cytokines, while simultaneously suppressing the expression of cell cycle-related genes. Mechanistically, we observed pervasive upstream translation in the 5' UTRs of cell cycle-related mRNAs, which contributes to cell cycle arrest during the early phase of inflammatory response. Notably, we identified a unique codon preference toward A/U in the third position of codons in macrophages, which contrasts with the G/C preference commonly observed in other tissues. AU codon preference increases the stability and translation efficiency of cell cycle-related mRNAs, promoting cell cycle restoration after extended LPS exposure. These findings reveal that uORF translation and codon usage bias are critical components of translational regulation during macrophage polarization, highlighting a potential therapeutic intervention for modulating immune activation via macrophage-specific codon optimization.

Animals↗

PubMed2026

Ribo-ITP enables identification of translons from limited input samples.

In the last decade, an unexpectedly large number of translated regions (translons) have been discovered using ribosome profiling and proteomics. Translons can act as regulatory elements or encode functional micropeptides. However, identification of translons has been limited to cell lines or large organs due to high input requirements for conventional ribosome profiling and mass spectrometry. Here, we address this input limitation using Ribo-ITP on difficult-to-collect samples such as microdissected hippocampal tissues and single preimplantation embryos to identify thousands of translons. To test the translational capacity of the identified translons, we engineer a translon-dependent GFP reporter system and detect expression of translons initiating at ATG and near-cognate start codons in mouse embryonic stem cells (mESCs). We identify distinct expression patterns of translons using a comparative analysis of more than a thousand ribosome profiling datasets across a wide range of cell types. Further, using a machine learning model, we predict that specific upstream translons in synaptically enriched mRNAs regulate translation efficiency of the annotated coding region. Taken together, we present a proof-of-concept study to identify non-canonical translation events from low input samples which can be applied to cell and tissue types inaccessible to conventional methods.

Animals↗

PubMed2024

Abundant mRNA m1A modification in dinoflagellates: a new layer of gene regulation.

Dinoflagellates, a class of unicellular eukaryotic phytoplankton, exhibit minimal transcriptional regulation, representing a unique model for exploring gene expression. The biosynthesis, distribution, regulation, and function of mRNA N1-methyladenosine (m1A) remain controversial due to its limited presence in typical eukaryotic mRNA. This study provides a comprehensive map of m1A in dinoflagellate mRNA and shows that m1A, rather than N6-methyladenosine (m6A), is the most prevalent internal mRNA modification in various dinoflagellate species, with an asymmetric distribution along mature transcripts. In Amphidinium carterae, we identify 6549 m1A sites characterized by a non-tRNA T-loop-like sequence motif within the transcripts of 3196 genes, many of which are involved in regulating carbon and nitrogen metabolism. Enriched within 3'UTRs, dinoflagellate mRNA m1A levels negatively correlate with translation efficiency. Nitrogen depletion further decreases mRNA m1A levels. Our data suggest that distinctive patterns of m1A modification might influence the expression of metabolism-related genes through translational control.

Dinoflagellida↗

PubMed1979

Wheat embryo ribonucleates. XIV. Mass isolation of mRNA from wheat germ and comparison of its translational capacity with that of mRNA from imbibing wheat embryos.

Commercially milled wheat germ is shown to be a convenient source material for facile recovery of mass (milligram) quantities of highly purified poly(A)-rich RNA. This poly(A)-rich RNA is efficiently translated in a nuclease-treated extract of rabbit reticulocytes. By sucrose density gradient fractionation of bulk poly(A)-rich RNA from wheat germ, it has been possible to show that there is a direct relationship between the molecular weights of the polypeptide products of cell-free synthesis and the molecular weights of the wheat mRNA molecules which program their synthesis. As assessed by SDS -- polyacrylamide gel electrophoresis, the same array of polypeptides is synthesized when nuclease-treated reticulocyte extract is programmed by poly(A)-rich RNA from either commercially supplied or laboratory-prepared wheat embryos. Significantly, there are gross quantitative if not qualitative differences between the translational capacities of poly(A)-rich RNA from dry and imbibing wheat embryos, and the possible importance of these differences for interpreting a changing pattern of polypeptide synthesis in imbibing wheat embryos is the subject of a brief discussion.

Animals↗

PubMed1979

Programmed appearance of translatable flagellar tubulin mRNA during cell differentiation in Naegleria.

The programmed de novo synthesis of flagellar tubulin during the hour-long differentiation of Naegleria gruberi from amoebae to flagellates is our paradigm for the study of gene expression during cell differentiation. This paper reports the efficient translation of flagellar tubulin mRNA in the wheat germ cell-free system directed by total or polyadenylated RNA extracted from differentiating cells. The tubulin in the in vitro product has a subunit molecular weight of 55,000, separates into alpha and beta subunits under suitable conditions of polyacrylamide gel electrophoreis and co-polymerizes with calf brain tubulin. At least half of the tubulin synthesized in vitro is precipitated by antibodies specific to flagellar tubulin, and the immunoprecipitated tubulin subunits yield peptide maps similar to those of outer doublet tublin. Flagellar tubulin is the predominant protein synthesized in the cell-free system, and amounts to about 5% of the polypeptides whose synthesis is directed by total RNA from differentiating cells. In contrast, little or no flagellar tubulin is synthesized when the cell-free system is directed by RNA extracted from amoebae prior to differentiation. Translation assays show that at least 92% of the flagellar tubulin mRNA appears during differentiation. The time course of appearance of this mRNA was measured by quantitative immunoprecipitation of the cell-free products. Under conditions where cells from flagella 60 min after initiation of differentiation, translatable flagellar tubulin mRNA was first detected at 20 min, reached a maximum at about 60 min and then declined. An excellent correlation was observed between the amount of translatable flagellar tubulin mRNA and the previously measured rates of flagellar tubulin synthesis in vivo. These results indicate that synthesis of flagellar tubulin is a direct reflection of the abundance of its mRNA, and provide the molecular techniques for dissection of the factors that regulate the rapid appearance of this structural protein during differentiation.

Animals↗

PubMed1979

Free cytoplasmic messenger ribonucleoprotein complexes from rabbit reticulocytes.

Free cytoplasmic globin mRNA containing mRNP-particles were isolated from rabbit reticulocytes by zonal sucrose gradient centrifugation and their properties were compared with mRNP particles isolated in the same way from EDTA-dissociated reticulocyte polyribosomes. The average poly(A)-length of 9S mRNA from free cytoplasmic mRNP was 17-20 nucleotides being about two times shorter than the average poly(A)-length of polysomal 9S mRNA. The protein composition of the free cytoplasmic mRNP particles disclosed the absence of the 76,000 dalton protein which is associated with the 3'poly(A)-segment of polysomal globin mRNA. It was concluded that free cytoplasmic mRNP-particles from rabbit reticulocytes can be classified as "old" mRNP in a post-translational phase. Free cytoplasmic mRNPs were translated in heterologous cell-free systems as well as in Xenopus laevis oocytes. Addition of hemin stimulated the synthesis of alpha-globin in all systems, while the presence of the cap analogue m7G(5')p inhibited translation of free cytoplasmic mRNA completely. The latter finding suggested that free cytoplasmic mRNA has a 5' terminal "cap". Shortening of the poly(A)-segment with concomitant loss of the 76,000 dalton protein may lead to less efficient translation of free cytoplasmic mRNP.

Animals↗

PubMed1977

Translation of Drosophila melanogaster sequences in Escherichia coli.

Thirty-seven independently cloned segments of Drosophila melanogaster DNA (Dm segments) were individually tested for their ability to promote the synthesis of new polypeptides in Escherichia coli K-12. The cloning vector was the pSC101 plasmid and the test system consisted of E. coli K-12 minicells that contained the hybrid pDm plasmids. Each of four pDm plasmids produced a new polypeptide, and one, pDm107, was selected for detailed mapping of the sequences required for the translation of its 38,000-dalton polypeptide, the Dm107 protein. Mapping was accomplished by constructing (i) deletion derivatives of pDm107 and (ii) new plasmids consisting of fragments of the Dm107 segment inserted into other vectors, and then testing these hybrids for their ability to promote the synthesis of the Dm107 protein, or truncated versions of this protein, in minicells. The 1000 base pairs of sequences that are translated to yield the Dm107 protein were thereby mapped at the center of the 18,000-base pair Dm107 segment, which consists of nonrepetitive sequences located at the base of the right arm of chromosome 2. The four polypeptides produced by the four pDm plasmids require sequences of 4000 base pairs for their translation, and the total amount of DNA in the 37 cloned Dm segments that were tested is approximately 400,000 base pairs. Because no new polypeptides were detected with the remaining 33 pDm plasmids, the fraction of D. melanogaster sequences that can be efficiently translated in E. coli K-12 is estimated to be 1 x 10(-2).

Animals↗

PubMed2026

A truncated COL10A1 protein causes Schmid metaphyseal chondrodysplasia via protein downregulation and impairing α1 trimer formation and secretion.

Schmid-type metaphyseal chondrodysplasia (SMCD) is primarily caused by mutations in the COL10A1 gene. This study reports a novel frameshift mutation, c.1940dup (p.Asn647Lysfs*2), identified in a Chinese SMCD pedigree. The mutation did not alter messenger RNA levels but significantly reduced COL10A1 protein expression. The mutant protein lacks the C-terminal 33 amino acids, resulting in a truncated polypeptide of 648 residues with a lower molecular weight than the wild-type protein. Degradation kinetics analysis showed no evidence of accelerated turnover. Notably, even under complete inhibition of degradation pathways, mutant protein expression remained substantially lower than that of wild-type, suggesting a potential defect in translational efficiency. Furthermore, the mutation severely disrupted the assembly of the characteristic collagen X trimer and led to markedly reduced extracellular secretion, as measured by accumulated protein levels in conditioned medium. These findings demonstrate that the c.1940dup mutation contributes to SMCD pathogenesis through coordinated mechanisms involving protein truncation, reduced expression, probable translational deficiency, and defective trimer formation and secretion, thereby revealing new potential therapeutic targets.

Osteochondrodysplasias↗

PubMed2026

Polysomal Profiling Coupled to Allele-Specific Proteomics Reveals an EIF4H TranSNP Allele Possessing Higher mRNA Translation Potential.

To search for genetic sources of allele-specific mRNA translation, we leveraged heterozygous polymorphisms and variants present in the exome of HCT116 colorectal adenocarcinoma-derived cells, computing allelic fractions from both total and polysome-associated RNA from RNA-Seq data. Allelic imbalance in polysomal RNA led us to nominate 52 coding variants associated with allele-specific mRNA translation, of which 16 are nonsynonymous. To validate instances of allele-specific translation, a proteomics workflow was developed that combines label-free shotgun analysis, high-pH reversed-phase peptide fractionation, and targeted parallel reaction monitoring using isotope-labeled peptide standards. Using this approach, we provide proof-of-concept validation of the heterozygous G>A, R183H missense single-nucleotide variant rs1554710467 in the eukaryotic initiation factor 4H (EIF4H) gene. The variant is present in two EIF4H alternatively spliced variants, which showed equivalent translation efficiency in HCT116 cells but differ in abundance. The alternative peptide containing H183 was significantly more abundant than the corresponding reference peptide containing R183, consistent with the over-representation of the alternative allele in polysomal RNA in HCT116 cells. A dual-fluorescence ribosome-stalling assay confirmed the enhanced translation potential of the variant allele. The two EIF4H allelic proteins exhibited similar stability and subpolysomal localization. This study demonstrates the feasibility of using allele-specific proteomics at the endogenous protein levels by exploiting heterozygous coding variants. Overall, our approach extends the toolbox available to investigate allele-specific differences in mRNA translation potential, a relatively underexplored layer of gene expression regulation that could reveal interindividual differences in disease-relevant phenotypes.

Humans↗

PubMed2026

mRNA therapy: A novel approach for retinal neurodegenerative diseases.

Retinal neurodegeneration remains a major cause of irreversible vision loss, yet current therapeutic options are limited in effectiveness. Although gene therapies have shown clinical potential, the overexpression platforms they rely on, such as adeno-associated virus DNA, are constrained by safety concerns, limited efficacy, and cargo size restrictions. In contrast, mRNA therapy has gained recognition as a compelling alternative, enabling rapid and efficient protein expression without the risk of genomic integration. This review synthesizes recent advances in mRNA engineering, delivery systems, and administration routes for retinal applications, and highlight strategies to enhance targeting, penetration, and controlled release through interdisciplinary collaboration between ophthalmology and bioengineering. In recent years, engineered mRNA formats, including chemically modified linear, circular, and self-amplifying RNA, can achieve higher translation efficiency within a tunable expression window. The transient nature and relatively low immunogenicity of in vitro transcribed mRNA support repeat dosing without insertional mutagenesis. Advances in nanocarriers, particularly lipid nanoparticles, have enabled preferential delivery to retinal neurons, Müller glia, and pigment epithelium via intraocular administration, while improving mRNA stability and transfection efficiency. In preclinical studies, mRNA has been widely used to deliver gene-editing tools, transcription factors, and supplementary functional proteins. In disease models such as optic nerve crush and laser-induced choroidal neovascularization, mRNA-based therapies enhance neuroprotection and suppress pathological angiogenesis in the injured retina, with favorable ocular safety profiles. However, it remains largely unexplored how the intrinsic advantages of mRNA therapy can be leveraged to develop tailored strategies for complex retinal disorders. Consistent with this gap, mRNA platforms have not yet been widely incorporated into retinal research or clinical practice. In parallel, clinical translation also lags: despite encouraging outcomes of lipid nanoparticle-mRNA formulations in preclinical models, no candidates have progressed into retinal clinical trials. This review draws on the complex pathology and therapeutic logic of retinal neurodegeneration. It proposes that mRNA therapy enables multitarget, repeatable, stage-specific interventions that align with the dynamic evolution of diseases and the requirements of combination therapy in retinal diseases. It may be used to support neuroprotection, axon regeneration, and neurovascular regulation. By integrating data across experimental models and modalities, this review outlines representative cases and experimental paradigms to guide rational trial design and carrier selection. Taken together, technical progress and evolving application strategies position mRNA therapy as a compelling therapeutic avenue for retinal neurodegeneration.

administration↗

PubMed1977

Translation in vitro of rat liver messenger RNA coding for ligandin (glutathione S-transferase B).

Poly(A)-containing rat liver mRNA isolated from animals injected with phenobarbital and uninjected controls was translated efficiently in a wheat-germ system. The synthesis of ligandin (glutathione S-transferase B; glutathione transferase; RX-gluathione R-transferase, EC 2.5.1.18) was detected by immunoprecipitation with a highly purified monospecific ligandin antibody and analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The extent of incorporation of [35S]methionine into ligandin in the translation system was similar for poly(A)-containing messages from un-infected animals and those treated with phenobarbital.

Animals↗

PubMed1979

Effect of benzo[a]pyrene-diolepoxide on infectivity and in vitro translation of phage MS2 RNA.

Previous studies have shown that alkylation of MS2 RNA by certain derivatives of polycyclic aromatic hydrocarbons renders it noninfectious. Since phage RNA serves as a template for translation and transcription, either of these RNA-directed processes, or both, could be responsible in vivo for the inhibition of phage replication by metabolically activated hydrocarbons. The present study correlates the degree of inhibition of MS2 RNA infectivity, at various levels of alkylation by (+/-)-trans, 7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzol[a]pyrene, with the translation efficiency in vitro of the same alkylated RNA for the synthesis of viral synthetase and of maturation and coat proteins. The results indicate that dihydroxyepoxy-tetrahydrobenzo[a]pyrene modification of MS2 RNA impairs its template capacity for the synthesis of phage-specific proteins; this inhibition is insufficient, however, to account for the loss of RNA infectivity at lower molar ratios of alkylation. For the three viral proteins synthesized in vitro, the translation of RNA synthetase is much more sensitive to MS2 RNA modification than either coat or maturation protein synthesis. Our results also indicate that the loss of viral RNA infectivity follows a single-hit inactivation mechanism, whereas several alkylation events in the viral RNA synthetase cistron may be necessary to block translation of this gene product.

Alkylation↗

PubMed1979

Alterations in translational control mechanisms in Friend erythroleukemic cells during DMSO induced differentiation.

Induction of erythroid differentiation in Friend erythroleukemia cells causes a reduction in the rate of protein synthesis and a prolonged growth cycle of the cells. A study of the relative translational efficiencies (RTE) of viral mRNAs and selected host cell mRNAs revealed unexpected low RTEs of the mRNA coding for the gp70 protein and RTE of the mRNA coding for the p30 protein. The RTEs of viral and of some host mRNAs show significant changes during differentiation. The rate of synthesis of corresponding proteins are altered non-coordinately, indicating transcriptional and/or posttranscriptional regulation of mRNA, as well as amplified regulation at the level of translation of mRNA.

Cell Differentiation↗