mRNA Template Research Articles

Translation of rod-like template sequences into homochiral assemblies of stacked helical oligomers.

At the molecular level, translation refers to the production of a new entity according to a template that has a different chemical composition. In this way, chemical information may be translated from one molecule into another. The process is useful to synthesise structures and thus functions that might be difficult to create otherwise and reaches exquisite levels of efficiency in biological systems as illustrated by protein expression from mRNA templates1,2 or by the assembly of the tobacco mosaic virus capsid protein according to the length of its RNA3. In synthetic systems, examples of template-directed syntheses are numerous4–6, but general and versatile schemes in which a non-natural sequence actually encodes the information necessary to produce a different sequence are few, and far from being optimised7–10. Here we show high-fidelity enzyme-free translation of long rod-like alkylcarbamate oligomers into well-defined sequences of stacked helical aromatic oligoamides. Features present in the rods, including the number and distance between carbamate functions and stereogenic centres, template the self-assembly of complementary stacks of helices each having a defined right (P) or left (M) handedness, length, and single or double helicity. This process enables the production of very large (> 20 kDa) abiotic artificial folded architectures (foldamers11) that may, for example, serve as scaffolds to organize appended functional features at positions in space defined with atomic precision across nanometric distances.

A resource dependent protein synthesis model for evaluating synthetic circuits

Reliable in silico design of synthetic gene networks necessitates novel approaches to model the process of protein synthesis under the influence of limited resources. We present such a novel protein synthesis model which originates from the Ribosome Flow Model and among other things describes the movement of RNA-polymerase and ribosomes on mRNA and DNA templates, respectively. By analyzing the convergence properties of this model based upon geometric considerations, we present additional insights into the dynamic mechanisms of the process of protein synthesis. Further, we demonstrate how this model can be used to evaluate the performance of synthetic gene circuits under different loading scenarios.

Modeling sRNA-Regulated Plasmid Maintenance.

We study a theoretical model for the toxin-antitoxin (hok/sok) mechanism for plasmid maintenance in bacteria. Toxin-antitoxin systems enforce the maintenance of a plasmid through post-segregational killing of cells that have lost the plasmid. Key to their function is the tight regulation of expression of a protein toxin by an sRNA antitoxin. Here, we focus on the nonlinear nature of the regulatory circuit dynamics of the toxin-antitoxin mechanism. The mechanism relies on a transient increase in protein concentration rather than on the steady state of the genetic circuit. Through a systematic analysis of the parameter dependence of this transient increase, we confirm some known design features of this system and identify new ones: for an efficient toxin-antitoxin mechanism, the synthesis rate of the toxin’s mRNA template should be lower that of the sRNA antitoxin, the mRNA template should be more stable than the sRNA antitoxin, and the mRNA-sRNA complex should be more stable than the sRNA antitoxin. Moreover, a short half-life of the protein toxin is also beneficial to the function of the toxin-antitoxin system. In addition, we study a therapeutic scenario in which a competitor mRNA is introduced to sequester the sRNA antitoxin, causing the toxic protein to be expressed.

Identification of a Novel MPL Loss of Function Mutation in a Patient with Cyclic Thrombocytopenia and Characterization of This Syndrome

Introduction: Cyclic thrombocytopenia (CT) is a rare disease characterized by periodic fluctuations in platelet counts. The etiology of this disease has not been fully elucidated. Here, we present molecular characterization of CT in a 53-year-old male patient who has platelet counts ranging between 1 x 109/L to >400 x 109/L during a cycle period of 40 days which has occurred over several years.Methods: Blood transcriptome profiles and plasma cytokine levels were investigated in 24 samples that were sequentially collected every 3-4 days to cover two complete cycles. Total RNA was extracted after lysing red blood cells. 3SEQ (3′-end RNA sequencing for expression quantification) was performed for transcriptome profiling. SAMSeq quantitative analyses were conducted to identify differentially expressed genes. Three screening criteria were applied to this data set to identify a panel of exclusive platelet-specific genes. Plasma TPO (thrombopoietin)/testosterone/ estradiol/CD41a/CD42b levels were detected by ELISA. A total of 62 cytokines were screened using a luminex immunoassay. MPL (TPO receptor) gene was Sanger-sequenced using blood DNA and mRNA templates. DNA was extracted from the patient’s hair follicles to exam the presence of the identified MPL mutation, and in-silico predictions were conducted to evaluate the MPL mutation. The wild-type (WT) and c.1210G>A mutant MPL expression constructs were generated and transfected into Ba/F3 cells for stable MPL expression. TPO-stimulated, IL-3 independent growth of MPL-expressing Ba/F3 cells were assessed to determine the function of MPL proteins.Results: Plasma TPO levels cycled between 6 to 2745 pg/mL during the sampling period; TPO levels inversely mirrored the fluctuation of platelet counts and preceded the latter by 3-4 days. Additional plasma cytokines demonstrated either an inverse correlation (e.g. FasL, GM-CSF, and TNF-β, etc.), or well synchronized correlation (e.g. BDNF, PDGF-BB, and RANTES etc.), with platelet counts. The patient was negative for platelet autoantibodies. Transcriptome analysis revealed 977 genes with expression that positively correlated with platelet count changes. Unsupervised clustering stratified these genes into subgroups, including: i) platelet-specific genes, which precede platelet count changes by 3-4 days; ii) platelet-modulated genes, which follow platelet count changes and are regulated by coagulation factors (e.g. F2) and platelet-contained growth factors (e.g. PDGF-BB and EGF); and iii) neutrophil-specific genes, which precede platelet count changes by 7-10 days. Among platelet-specific genes, 34 genes were identified to have >50-fold induction over a cycle, and are present in the platelet transcriptome [Rowley JW, 2011]. A heat map of these exclusive platelet genes is shown in Figure 1A.A novel MPL c.1210G>A heterozygous mutation, which leads to a p.Gly404Arg substitution, was identified in both blood and hair follicle DNA samples of the patient. This substitution resides in a highly conserved site and is predicted to be deleterious. In addition, the mutation creates a novel splice site, which results in the insertion of a 36 bp intron fragment in approximately 15% of MPL transcripts. In vitro analysis confirmed that MPL c.1210G>A is a loss-of-function (LOF) variant. Ba/F3 cells that express WT MPL exhibited IL-3 independent growth in the presence of TPO; however, the Gly404Arg mutant failed to support TPO-stimulated growth (Figure 1B).Conclusion: We describe a patient with CT who exhibits fluctuations of multiple cytokines including an inverse correlation between TPO and platelet counts, and profound gene expression changes in neutrophil- and platelet-specific gene expressions which precede platelet count fluctuations. These data suggest that the cyclic megakaryopoiesis and thrombopoiesis underlies pathogenesis of the disease. Moreover, the identified novel LOF MPL allele may contribute to the disease pathogenesis. The biologic and molecular annotation of this case presents a unique opportunity to examine the transcriptional regulation of platelet homeostasis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An Accurate, Simple, and Inexpensive Assay to Diagnose F8 Gene Inversion Mutations in Hemophilia a Patients and Carriers

The most frequent mutations resulting in hemophilia A are an intron 22 or intron 1 F8 gene inversion, which together result in ~50% of severe hemophilia A cases. We report simple and accurate RNA- and DNA-based assays to detect these mutations in patients and carrier mothers. They do not require specialized equipment or expensive reagents, and therefore they may provide useful and economic protocols that could be standardized for central laboratory testing.RNA is purified from a whole-blood RNA-stabilized sample or from peripheral blood mononuclear cells, and a two-step nested PCR (RT-PCR followed by PCR) reaction is carried out to amplify complementary DNA (cDNA) fragments with F8 sequences spanning the exon 22-23 splice site (intron-22 inversion test) or the exon 1-2 splice site (intron-1 inversion test). These sequences will be amplified from F8 mRNA without an intron-22 or intron-1 inversion mutation, respectively. Additional RT-PCR reactions are carried out to amplify a unique intron 22 inverted-gene mRNA sequence extending from F8 exon 19 to the first in-frame stop codon within intron 22, or a chimeric intron 1 inverted-gene transcript containing sequences from F8 exon 1 and the adjacent VBP1 gene. These latter two DNA products will be amplified only from F8 mRNA isolated from patients with an intron-22 or an intron-1 inversion mutation, respectively. Amplification of a FVIII C2 domain sequence serves as an internal control for the RT-PCR reactions, as this product will be generated from samples with and without an inversion mutation. Primers were designed to amplify PCR products having different lengths (between 200 and 600 bp) that could be clearly distinguished following electrophoresis on agarose gels. Samples from carrier mothers amplified the normal control as well as the inversion-mutation-specific bands, as expected.A nested PCR protocol reliably produced interpretable results from low template mRNA concentrations, while a one-step multiplexed RT-PCR version of the assay produced the expected bands, with fewer experimental steps, from higher template mRNA concentrations.The standard DNA-based inverse-shifting PCR (IS-PCR) reaction may be used for primary diagnosis and/or to classify intron 22 inversions as type 1 or type 2 (reflecting the specific homologous recombination site). We utilized a commercially available Bcl I enzyme that efficiently cleaves DNA in 5 minutes, a substantial improvement over previous IS-PCR protocols that required overnight DNA digests.These rapid, accurate protocols can economically detect or rule F8 inversion mutations via same-day testing of patient samples, providing timely information that is useful to families and in clinical evaluations. DisclosuresDutta:Grifols, Inc: Research Funding. Ragni:OPKO: Research Funding; Vascular Medicine Institute: Research Funding; Tacere Benitec: Consultancy; SPARK: Research Funding; Shire: Consultancy; Novo Nordisk: Research Funding; Genentech: Research Funding; CSL Behring: Research Funding; Biomarin: Consultancy; Biogen: Consultancy, Research Funding; Baxalta: Research Funding; Alnylam Pharmaceuticals: Consultancy, Research Funding.

Ratio of T-Helper Type 1 (Th1) to Th17 Cytokines in Whole Blood Is Associated With Human β-Defensin 3 Expression in Skin and Persistent Staphylococcus aureus Nasal Carriage.

Nasal colonization has gained attention as an effect modifier in Staphylococcus aureus vaccine trials, suggesting interference of carriage with T-cell immunity. Likewise, T-cell signals may be involved in regulating effectors of epithelial innate defense. Whole blood from 43 persistent carriers and 49 noncarriers was stimulated with viable S. aureus T-helper type 1 (Th1), Th2, and Th17 cytokine expression was measured, compared between carrier groups, and linked with data on human β-defensin 3 (hBD-3) messenger RNA (mRNA) in skin while adjusting for transcriptionally relevant promoter haplotypes. Compared with carriers, stimulated whole blood from noncarriers contained on average 60% more interferon γ mRNA (P = .031) and 19% less interleukin 17A (IL-17A) mRNA (P = .11), resulting in, on average, a 90% higher IFN-γ to IL-17A mRNA ratio (P = .003). In a multivariable model, per duplication of the mRNA template, the risk of being a carrier increased by 93% for IL-17A (odds ratio [OR], 1.93; 95% confidence interval [CI], 1.10-3.41; P = .023) and decreased by 35% for IFN-γ (OR, 0.65; 95% CI, 0.47-0.91; P = .01). Independent of carriage and DEFB promotor haplotype, a 1-unit increase in the IFN-γ to IL-17A mRNA ratio (mean ± SD, 5.93 ± 1.60) led to a 24% increase in hBD-3 transcription in experimentally wounded human skin (P = .003). A low Th1 to Th17 mRNA ratio increases the propensity for persistent S. aureus nasal colonization, with downregulated hBD-3 transcription providing a potential link.

A microchip for integrated single-cell genotoxicity assay

With the development of large-scale biologic databases, precision medicine is becoming a frontier in biomedical research. As a main focus of precision medicine study, cancer has been widely accepted as a disease born out of inherited genetic variations or accumulating genomic damage. At the single-cell level, microfluidics or lab-on-a-chip technology for cancer study is an emerging tool for improving risk assessment, diagnostic categories and therapeutic strategies. This work presents a multi-layer microchip for single-cell gene expression profiling. Treated by three drug reagents (i.e. methyl methanesulfonate, docetaxel and colchicine) with varied concentrations and time lengths, individual human breast cancer cells (MCF-7) are then lysed on-chip, and the released mRNA templates are captured and reversely transcribed into cDNA on microbead surface. Three genes (GAPDH, CDKN1A, AURKA) are amplified and quantified simultaneously through triplex real-time polymerase chain reactions (qPCR). Readout per run is set to be eighteen, and can be further improved following same approach. The microchip is able to integrate all steps of single-cell gene expression profiling, and provide precision study of drug induced genotoxicity with reduced reagents consumption per reaction and instrumental cost.

A Microchip for Integrated Single-Cell Gene Expression Profiling and Genotoxicity Detection.

Microfluidics-based single-cell study is an emerging approach in personalized treatment or precision medicine studies. Single-cell gene expression holds a potential to provide treatment selections with maximized efficacy to help cancer patients based on a genetic understanding of their disease. This work presents a multi-layer microchip for single-cell multiplexed gene expression profiling and genotoxicity detection. Treated by three drug reagents (i.e., methyl methanesulfonate, docetaxel and colchicine) with varied concentrations and time lengths, individual human cancer cells (MDA-MB-231) are lysed on-chip, and the released mRNA templates are captured and reversely transcribed into single strand DNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclin-dependent kinase inhibitor 1A (CDKN1A), and aurora kinase A (AURKA) genes from single cells are amplified and real-time quantified through multiplex polymerase chain reaction. The microchip is capable of integrating all steps of single-cell multiplexed gene expression profiling, and providing precision detection of drug induced genotoxic stress. Throughput has been set to be 18, and can be further increased following the same approach. Numerical simulation of on-chip single cell trapping and heat transfer has been employed to evaluate the chip design and operation.

Slip of grip of a molecular motor on a crowded track: Modeling shift of reading frame of ribosome on RNA template

We develop a stochastic model for the programmed frameshift of ribosomes synthesizing a protein while moving along a mRNA template. Normally the reading frame of a ribosome decodes successive triplets of nucleotides on the mRNA in a step-by-step manner. We focus on the programmed shift of the ribosomal reading frame, forward or backward, by only one nucleotide which results in a fusion protein; it occurs when a ribosome temporarily loses its grip to its mRNA track. Special “slippery” sequences of nucleotides and also downstream secondary structures of the mRNA strand are believed to play key roles in programmed frameshift. Here we explore the role of an hitherto neglected parameter in regulating −1 programmed frameshift. Specifically, we demonstrate that the frameshift frequency can be strongly regulated also by the density of the ribosomes, all of which are engaged in simultaneous translation of the same mRNA, at and around the slippery sequence. Monte Carlo simulations support the analytical predictions obtained from a mean-field analysis of the stochastic dynamics.

A multi-layer microchip for high-throughput single-cell gene expression profiling

Microfluidics or Bio-MEMS technology offers significant advantages for performing high-throughput screens and sensitive assays. The ability to correlate single-cell genetic information with cellular phenotypes is of great importance to biology and medicine because it holds the potential to gain insight into disease pathways that is unavailable from ensemble measurements. Previously, we reported two kinds of prototypes for integrated on-chip gene expression profiling at the single-cell level, and the throughput was designed to be 6. In this work, we present a five-layer microfluidic system for parallelized, rapid, quantitative analysis of RNA templates with low abundance at the single-cell level. The microchip contains two multiplexors and one partitioning valve group, and it leverages a matrix (6 × 8) of quantitative reverse transcription polymerase chain reaction (RT–qPCR) units formed by a set of parallel microchannels concurrently controlled by elastomeric pneumatic valves, thereby enabling parallelized handling and processing of biomolecules in a simplified operation procedure. A comprehensive metallic nanofilm with passivation layer is used to run polymerase chain reaction (PCR) temperature cycles. To demonstrate the utility of the approach, artificial synthesized RNA templates (XenoRNA) and mRNA templates from single cells are employed to perform the 48-readout RT–qPCRs. The PCR products are imaged on a fluorescence microscope using a hydrolysis probe/primer set (TaqMan). Fluorescent intensities of passive reference dye and a fluorescein amidite reporter dye are acquired and measured at the end of PCR cycles.

37. Towards Clinical Translation of Hematopoietic Stem Cell Gene Editing for the Correction of SCID-X1 Mutations

The scope of genetic engineering of hematopoietic stem/progenitor cells (HSPC) has broadened from gene replacement to targeted genome editing using a choice of artificial nucleases, which enables precise modifications of endogenous genes. We recently showed that gene editing in the primitive HSPC is constrained by gene transfer efficiency and limited proficiency of homology directed DNA repair. By combining Integrase Defective Lentiviral Vectors (IDLV) for donor template delivery and mRNA transfection for nucleases expression and tailoring culture conditions, we overcame in part these barriers and provided evidence of increased targeted integration (TI) in human HSPC. We achieved TI of a corrective cDNA into a mutational hotspot of IL2RG gene in long-term repopulating HSPC with efficiency and specificity potentially suitable for clinical translation. Here, in order to improve the tolerability of the procedure and establish a transferable-to-the-clinic gene correction protocol we optimized reagents and scaled-up the gene editing procedure. We developed new ZFNs targeting the upstream region of the IL2RG gene to correct the majority of SCID-X1 mutations with only one ZFN/donor set. By targeting a corrective cDNA into intron-1 of IL2RG gene in primary T cells, we found that targeted cells were functionally indistinguishable from wild-type ones, proving the functionality of the edited gene. We compared the performance of IDLV and AAV6 as donor vehicles for HSPC targeting and found that both vectors are similarly proficient in the delivery of donor templates for HDR, allowing up to 20% TI in bulk HSPC. To improve nuclease expression while decreasing cellular innate response to mRNA transfection we included modified nucleotides during mRNA production and performed HPLC purification after in-vitro transcription. The use of this optimized mRNA and clinical grade purification of IDLV (based on DNA removal followed by anion exchange chromatography) allowed decreasing type-1 interferon activation and electroporation toxicity, respectively. To further optimize ex-vivo HSPC manipulation we tested pyrimidoindole derivatives added to the culture and found a combination promoting HSPC expansion in conditions that preserve their primitive phenotype, increasing the yield of edited cells that are able to repopulate NSG mice. Finally, we demonstrated the therapeutic potential of our strategy by correcting the IL2RG gene in HSPC from a genotyped SCID-X1 patient. Currently, we are processing large scale lots of gene corrected cells using a high volume electroporator and have successfully treated up to 40 million of HSPCs. We envisage early clinical translation of this approach, which may benefit SCID-X1 patients while avoiding the risks of allogeneic transplantation or random transgene integration mediated by HSPC gene therapy with conventional integrating vectors.

Cooperation between the chloroplast psbA 5'-untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region.

Chloroplast mRNA translation is regulated by the 5'-untranslated region (5'-UTR). Chloroplast 5'-UTRs also support translation of the coding regions of heterologous genes. Using an in vitro translation system from tobacco chloroplasts, we detected no translation from a human immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5'-UTR. This lack of apparent translation could have been due to rapid degradation of mRNA templates or synthesized protein products. Replacing the psbA 5'-UTR with the E. coli phage T7 gene 10 5'-UTR, a highly active 5'-UTR, and substituting synonymous codons led to some translation of the tat coding region. The Tat protein thus synthesized was stable during translation reactions. No significant degradation of the added tat mRNAs was observed after translation reactions. These results excluded the above two possibilities and confirmed that the tat coding region prevented its own translation. The tat coding region was then fused to the psbA 5'-UTR with a cognate 5'-coding segment. Significant translation was detected from the tat coding region when fused after 10 or more codons. That is, translation could be initiated from the tat coding region once translation had started, indicating that the tat coding region inhibits translational initiation but not elongation. Hence, cooperation/compatibility between the 5'-UTR and its coding region is important for translational initiation.

Programmed −1 Frameshift of a Ribosome: Non-Monotonic Variation of Frameshift Efficiency with Increasing Stiffness of mRNA Secondary Structure

A ribosome is a molecular motor [1] that synthesises a polypeptide using a mRNA template that also serves as a track for its motor-like directed movement in steps of three nucleotides (a codon). However, on some special “slippery” sequence of nucleotides on the mRNA the reading frame of a ribosome can shift backward by one nucleotide which is identified as a −1 programmed frameshift. In addition to the slippery sequence, a secondary structure of the mRNA strand (for example, a pseudo knot), located downstream from the slippery sequence, is also essential for programmed −1 frameshift. Since the shifted reading frame picks up a sequence of amino acids that is, in general, different from the sequence that would have been selected by the unshifted original reading frame, a ribosome that resumes protein synthesis after such a frameshift produces a fusion protein. Often many ribosomes simultaneously move along the same mRNA track, all synthesising identical polypeptides. The stiffness of the mRNA secondary structure determines the extent of ribosomal crowding in the mRNA segment upstream from that structure. Using a theoretical model we demonstrate how the increasing stiffness of the secondary structure of the mRNA causes increasing crowding of the ribosomes that, in turn, leads to a non-monotonic variation of the efficiency of frameshift [3]. Our numerical estimates indicate that the novel phenomenon predicted by our theoretical model can be tested experimentally by ribosome profiling technique [4]. [1] D. Chowdhury, Biophys. J. 104, 2331 (2013). [2] J.F. Atkins and R.F. Gesteland (eds.) Recoding: Expansion of Decoding Rules Enriches Gene Expression, (Springer, 2010). [3] B. Mishra and D. Chowdhury, to be published (2015). [4] N.T. Ingolia, Nat. Rev. Genet. 15, 205 (2014).

Hippuristanol - A potent steroid inhibitor of eukaryotic initiation factor 4A.

Protein synthesis and its regulatory signaling pathways play essential roles in the initiation and maintenance of the cancer phenotype. Insight obtained over the last 3 decades on the mechanisms regulating translation in normal and transformed cells have revealed that perturbed control in cancer cells may offer an Achilles' heel for the development of novel anti-neoplastic agents. Several small molecule inhibitors have been identified and characterized that target translation initiation - more specifically, the rate-limiting step where ribosomes are recruited to mRNA templates. Among these, hippuristanol, a polyhydroxysteroid from the gorgonian Isis hippuris has been found to inhibit translation initiation by blocking the activity of eukaryotic initiation factor (eIF) 4A, an essential RNA helicase involved in this process. Herein, we highlight the biological properties of this compound, its potential development as an anti-cancer agent, and its use to validate eIF4A as an anti-neoplastic target.

Translation Inhibition by Rocaglates Is Independent of eIF4E Phosphorylation Status

Rocaglates are natural products that inhibit protein synthesis in eukaryotes and exhibit antineoplastic activity. In vitro biochemical assays, affinity chromatography experiments coupled with mass spectrometry analysis, and in vivo genetic screens have identified eukaryotic initiation factor (eIF) 4A as a direct molecular target of rocaglates. eIF4A is the RNA helicase subunit of eIF4F, a complex that mediates cap-dependent ribosome recruitment to mRNA templates. The eIF4F complex has been implicated in tumor initiation and maintenance through elevated levels or increased phosphorylation status of its cap-binding subunit, eIF4E, thus furthering the interest toward developing rocaglates as antineoplastic agents. Recent experiments have indicated that rocaglates also interact with prohibitins 1 and 2, proteins implicated in c-Raf-MEK-ERK signaling. Because increased ERK signaling stimulates eIF4E phosphorylation status, rocaglates are also expected to inhibit eIF4E phosphorylation status, a point that has not been thoroughly investigated. It is currently unknown whether the effects on translation observed with rocaglates are solely through eIF4A inhibition or also a feature of blocking eIF4E phosphorylation. Here, we show that rocaglates inhibit translation through an eIF4E phosphorylation-independent mechanism.

Engineering WT1-Encoding mRNA to Increase Translational Efficiency in Dendritic Cells.

Dendritic cells (DCs) are the orchestrators of the immune system and are frequently used in clinical trials in order to boost the immune system in cancer patients. Among several available techniques for DC modification, mRNA electroporation is an interesting technique due to the favorable characteristics of mRNA. Antigen expression level and duration can be increased by multiple optimizations of an antigen-encoding mRNA template. Here, we describe different molecular modifications to a WT1-encoding mRNA construct in order to increase antigen expression and the subsequent introduction of mRNA into DCs.

Polyethylenimine-based polyplex delivery of self-replicating RNA vaccines

Self-amplifying replicon RNA (RepRNA) are large molecules (12-14kb); their self-replication amplifies mRNA template numbers, affording several rounds of antigen production, effectively increasing vaccine antigen payloads. Their sensitivity to RNase-sensitivity and inefficient uptake by dendritic cells (DCs) – absolute requirements for vaccine design – were tackled by condensing RepRNA into synthetic, nanoparticulate, polyethylenimine (PEI)-polyplex delivery vehicles. Polyplex-delivery formulations for small RNA molecules cannot be transferred to RepRNA due to its greater size and complexity; the N:P charge ratio and impact of RepRNA folding would influence polyplex condensation, post-delivery decompaction and the cytosolic release essential for RepRNA translation. Polyplex-formulations proved successful for delivery of RepRNA encoding influenza virus hemagglutinin and nucleocapsid to DCs. Cytosolic translocation was facilitated, leading to RepRNA translation. This efficacy was confirmed in vivo, inducing both humoral and cellular immune responses. Accordingly, this paper describes the first PEI-polyplexes providing efficient delivery of the complex and large, self-amplifying RepRNA vaccines. From the Clinical EditorThe use of self-amplifying replicon RNA (RepRNA) to increase vaccine antigen payloads can potentially be useful in effective vaccine design. Nonetheless, its use is limited by the degradation during the uptake process. Here, the authors attempted to solve this problem by packaging RepRNA using polyethylenimine (PEI)-polyplex delivery vehicles. The efficacy was confirmed in vivo by the appropriate humoral and cellular immune responses. This novel delivery method may prove to be very useful for future vaccine design.

The Dedicated Chaperone Acl4 Escorts Ribosomal Protein Rpl4 to Its Nuclear Pre-60S Assembly Site.

Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent C-terminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S assembly site in the nucleus.

Interplay between the alpharetroviral Gag protein and SR proteins SF2 and SC35 in the nucleus.

Retroviruses are positive-sense, single-stranded RNA viruses that reverse transcribe their RNA genomes into double-stranded DNA for integration into the host cell chromosome. The integrated provirus is used as a template for the transcription of viral RNA. The full-length viral RNA can be used for the translation of the Gag and Gag-Pol structural proteins or as the genomic RNA (gRNA) for encapsidation into new virions by the Gag protein. The mechanism by which Gag selectively incorporates unspliced gRNA into virus particles is poorly understood. Although Gag was previously thought to localize exclusively to the cytoplasm and plasma membrane where particles are released, we found that the Gag protein of Rous sarcoma virus, an alpharetrovirus, undergoes transient nuclear trafficking. When the nuclear export signal of RSV Gag is mutated (Gag.L219A), the protein accumulates in discrete subnuclear foci reminiscent of nuclear bodies such as splicing speckles, paraspeckles, and PML bodies. In this report, we observed that RSV Gag.L219A foci appeared to be tethered in the nucleus, partially co-localizing with the splicing speckle components SC35 and SF2. Overexpression of SC35 increased the number of Gag.L219A nucleoplasmic foci, suggesting that SC35 may facilitate the formation of Gag foci. We previously reported that RSV Gag nuclear trafficking is required for efficient gRNA packaging. Together with the data presented herein, our findings raise the intriguing hypothesis that RSV Gag may co-opt splicing factors to localize near transcription sites. Because splicing occurs co-transcriptionally, we speculate that this mechanism could allow Gag to associate with unspliced viral RNA shortly after its transcription initiation in the nucleus, before the viral RNA can be spliced or exported from the nucleus as an mRNA template.

Codon Usage Influences the Local Rate of Translation Elongation to Regulate Co-translational Protein Folding

Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and has been proposed to regulate translation efficiency, accuracy, and protein folding based on the assumption that codon usage affects translation dynamics. The roles of codon usage in translation, however, are not clear and have been challenged by recent ribosome profiling studies. Here we used a Neurospora cell-free translation system to directly monitor the velocity of mRNA translation. We demonstrated that the preferred codons enhance the rate of translation elongation, whereas non-optimal codons slow elongation. Codon usage also controls ribosome traffic on mRNA. These conclusions were supported by ribosome profiling results invitro and invivo with template mRNAs designed to increase the signal-to-noise ratio. Finally, we demonstrate that codon usage regulates protein function byaffecting co-translational protein folding. These results resolve a long-standing fundamental question and suggest the existence of a codon usage code for protein folding.