Untranslated Leader Sequences and Enhanced Messenger RNA Translational Efficiency

The use of messenger RNA (mRNA) in gene therapy is increasing in recent years, due to its unique features compared to plasmid DNA: Transient expression, no need to enter into the nucleus and no risk of insertional mutagenesis. Nevertheless, the clinical application of mRNA as a therapeutic tool is limited by its instability and ability to activate immune responses; hence, mRNA chemical modifications together with the design of suitable vehicles result essential. This manuscript includes a revision of the strategies employed to enhance in vitro transcribed (IVT) mRNA functionality and efficacy, including the optimization of its stability and translational efficiency, as well as the regulation of its immunostimulatory properties. An overview of the nanosystems designed to protect the mRNA and to overcome the intra and extracellular barriers for successful delivery is also included. Finally, the present and future applications of mRNA nanomedicines for immunization against infectious diseases and cancer, protein replacement, gene editing, and regenerative medicine are highlighted.

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Ribosome recycling induces optimal translation rate at low ribosomal availability

Journal of The Royal Society Interface ◽

10.1098/rsif.2014.0589 ◽

2014 ◽

Vol 11 (98) ◽

pp. 20140589 ◽

Cited By ~ 21

Author(s):

E. Marshall ◽

I. Stansfield ◽

M. C. Romano

Keyword(s):

Protein Synthesis ◽

Messenger Rna ◽

Lattice Models ◽

Cellular Protein ◽

Current Phase ◽

Recycling Rate ◽

Translational Efficiency ◽

Loop Model ◽

Initiation Rate ◽

Translation Rate

During eukaryotic cellular protein synthesis, ribosomal translation is made more efficient through interaction between the two ends of the messenger RNA (mRNA). Ribosomes reaching the 3′ end of the mRNA can thus recycle and begin translation again on the same mRNA, the so-called ‘closed-loop’ model. Using a driven diffusion lattice model of translation, we study the effects of ribosome recycling on the dynamics of ribosome flow and density on the mRNA. We show that ribosome recycling induces a substantial increase in ribosome current. Furthermore, for sufficiently large values of the recycling rate, the lattice does not transition directly from low to high ribosome density, as seen in lattice models without recycling. Instead, a maximal current phase becomes accessible for much lower values of the initiation rate, and multiple phase transitions occur over a wide region of the phase plane. Crucially, we show that in the presence of ribosome recycling, mRNAs can exhibit a peak in protein production at low values of the initiation rate, beyond which translation rate decreases. This has important implications for translation of certain mRNAs, suggesting that there is an optimal concentration of ribosomes at which protein synthesis is maximal, and beyond which translational efficiency is impaired.

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RNA-Based Vaccines in Cancer Immunotherapy

Journal of Immunology Research ◽

10.1155/2015/794528 ◽

2015 ◽

Vol 2015 ◽

pp. 1-9 ◽

Cited By ~ 60

Author(s):

Megan A. McNamara ◽

Smita K. Nair ◽

Eda K. Holl

Keyword(s):

Immune Response ◽

Cancer Immunotherapy ◽

Rna Structure ◽

Messenger Rna ◽

Host Cells ◽

Translational Efficiency ◽

Tumor Associated Antigen ◽

Antigen Presenting ◽

Template Dna

RNA vaccines traditionally consist of messenger RNA synthesized byin vitrotranscription using a bacteriophage RNA polymerase and template DNA that encodes the antigen(s) of interest. Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm and then the resulting antigens are presented to antigen presenting cells to stimulate an immune response. Alternatively, dendritic cells can be loaded with either tumor associated antigen mRNA or total tumor RNA and delivered to the host to elicit a specific immune response. In this review, we will explain why RNA vaccines represent an attractive platform for cancer immunotherapy, discuss modifications to RNA structure that have been developed to optimize mRNA vaccine stability and translational efficiency, and describe strategies for nonviral delivery of mRNA vaccines, highlighting key preclinical and clinical data related to cancer immunotherapy.

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SPECIFIC PROTEIN SYNTHESIS IN CELLULAR DIFFERENTIATION

The Journal of Cell Biology ◽

10.1083/jcb.55.3.653 ◽

1972 ◽

Vol 55 (3) ◽

pp. 653-680 ◽

Cited By ~ 80

Author(s):

M. Paul ◽

M. R. Goldsmith ◽

J. R. Hunsley ◽

F. C. Kafatos

Keyword(s):

Protein Synthesis ◽

Messenger Rna ◽

Developmental Stages ◽

Specific Protein ◽

Chain Elongation ◽

Synthetic Activity ◽

Translational Efficiency ◽

Kinetics Of

Silkmoth follicles, arranged in a precise developmental sequence within the ovariole, yield pure and uniform populations of follicular epithelial cells highly differentiated for synthesis of the proteinaceous eggshell (chorion). These cells can be maintained and labeled efficiently in organ culture; their in vitro (and cell free) protein synthetic activity reflects their activity in vivo. During differentiation the cells undergo dramatic changes in protein synthesis. For 2 days the cells are devoted almost exclusively to production of distinctive chorion proteins of low molecular weight and of unusual amino acid composition. Each protein has its own characteristic developmental kinetics of synthesis. Each is synthesized as a separate polypeptide, apparently on monocistronic messenger RNA (mRNA), and thus reflects the expression of a distinct gene. The rapid changes in this tissue do not result from corresponding changes in translational efficiency. Thus, the peptide chain elongation rate is comparable for chorion and for proteins synthesized at earlier developmental stages (1.3–1.9 amino acids/sec); moreover, the spacing of ribosomes on chorion mRNA (30–37 codons per ribosome) is similar to that encountered in other eukaryotic systems.

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mRNA destabilisation through CDS-targeting is the primary role of endogenous miRNA in the green alga Chlamydomonas

10.1101/088807 ◽

2016 ◽

Cited By ~ 3

Author(s):

Betty Y-W. Chung ◽

Michael J. Deery ◽

Arnoud J. Groen ◽

Julie Howard ◽

David Baulcombe

Keyword(s):

Gene Expression ◽

Green Alga ◽

Messenger Rna ◽

Global Analysis ◽

Translational Efficiency ◽

Primary Role ◽

Regulate Gene Expression ◽

Coding Regions ◽

Unicellular Green Alga ◽

Regulate Gene

AbstractMicroRNAs regulate gene expression as part of the RNA-induced silencing complex, where the sequence identity of the miRNA provides the specificity to the target messenger RNA, and the result is target repression. The mode of repression can be through target cleavage, RNA destabilization and/or decreased translational efficiency. Here, we provide a comprehensive global analysis of the evolutionarily distant unicellular green alga Chlamydomonas reinhardtii to quantify the effects of miRNA on protein synthesis and RNA abundance. We show that, similar to metazoan systems, miRNAs in Chlamydomonas regulate gene-expression primarily by destabilizing mRNAs. However, unlike metazoan miRNA where target site utilization localizes mainly to 3’UTRs, in Chlamydomonas utilized target sites lie predominantly within coding regions. These results demonstrate that destabilization of mRNA is the main evolutionarily conserved mode of action for miRNAs, but details of the mechanism diverge between plant and metazoan kingdoms.

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A low-complexity region in human XRN1 directly recruits deadenylation and decapping factors in 5′–3′ messenger RNA decay

Nucleic Acids Research ◽

10.1093/nar/gkz633 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9282-9295 ◽

Cited By ~ 3

Author(s):

Chung-Te Chang ◽

Sowndarya Muthukumar ◽

Ramona Weber ◽

Yevgen Levdansky ◽

Ying Chen ◽

...

Keyword(s):

Messenger Rna ◽

Low Complexity ◽

Ribosome Profiling ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Translational Efficiency ◽

Mrna Levels ◽

Null Cell ◽

Integrated Network

Abstract XRN1 is the major cytoplasmic exoribonuclease in eukaryotes, which degrades deadenylated and decapped mRNAs in the last step of the 5′–3′ mRNA decay pathway. Metazoan XRN1 interacts with decapping factors coupling the final stages of decay. Here, we reveal a direct interaction between XRN1 and the CCR4–NOT deadenylase complex mediated by a low-complexity region in XRN1, which we term the ‘C-terminal interacting region’ or CIR. The CIR represses reporter mRNA deadenylation in human cells when overexpressed and inhibits CCR4–NOT and isolated CAF1 deadenylase activity in vitro. Through complementation studies in an XRN1-null cell line, we dissect the specific contributions of XRN1 domains and regions toward decay of an mRNA reporter. We observe that XRN1 binding to the decapping activator EDC4 counteracts the dominant negative effect of CIR overexpression on decay. Another decapping activator PatL1 directly interacts with CIR and alleviates the CIR-mediated inhibition of CCR4–NOT activity in vitro. Ribosome profiling revealed that XRN1 loss impacts not only on mRNA levels but also on the translational efficiency of many cellular transcripts likely as a consequence of incomplete decay. Our findings reveal an additional layer of direct interactions in a tightly integrated network of factors mediating deadenylation, decapping and 5′–3′ exonucleolytic decay.

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Regulation of HSP70 synthesis by messenger RNA degradation.

Cell Regulation ◽

10.1091/mbc.1.1.135 ◽

1989 ◽

Vol 1 (1) ◽

pp. 135-149 ◽

Cited By ~ 74

Author(s):

R B Petersen ◽

S Lindquist

Keyword(s):

Heat Shock ◽

Messenger Rna ◽

Untranslated Region ◽

Translation Termination ◽

Normal Growth ◽

Rna Degradation ◽

Translational Efficiency ◽

Hsp70 Mrna ◽

Drosophila Cells ◽

Hsp70 Synthesis

When Drosophila cells are heat shocked, hsp70 messenger RNA (mRNA) is stable and is translated at high efficiencies. During recovery from heat shock, hsp70 synthesis is repressed and its messenger RNA (mRNA) is degraded in a highly regulated fashion. Dramatic differences in the timing of repression and degradation are observed after heat treatments of different severities. The 3' untranslated region (UTR) of the hsp70 mRNA was sufficient to transfer this regulated degradation to heterologous mRNAs. Altering the translational efficiency of the message or changing its natural translation-termination site did not alter its pattern of regulation, although in some cases it changed the absolute rate of degradation. We have previously shown that hsp70 mRNA is very unstable when it is expressed at normal growth temperatures (from a metallothionein promoter). We report here that the 3' untranslated region of the hsp70 mRNA is responsible for this instability as well. We postulate that a mechanism for degrading hsp70 mRNA pre-exists in Drosophila cells, that it is inactivated by heat shock and that it is the reactivation of this mechanism that is responsible for hsp70 repression during recovery. This degradation system may be the same as that used by other unstable mRNAs.

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High performance Nanogold™-in situ hybridzation and its use in the detection of hybridized and PCR-amplified microscopical preparations

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166944 ◽

1996 ◽

Vol 54 ◽

pp. 896-897

Author(s):

G. W. Hacker ◽

I. Zehbe ◽

J. Hainfeld ◽

A.-H. Graf ◽

C. Hauser-Kronberger ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Messenger Rna ◽

High Performance ◽

Detection System ◽

Direct Methods ◽

Genetic Alterations ◽

Chain Reaction ◽

Protein Encoding ◽

Polymerase Chain

In situ hybridization (ISH) with biotin-labeled probes is increasingly used in histology, histopathology and molecular biology, to detect genetic nucleic acid sequences of interest, such as viruses, genetic alterations and peptide-/protein-encoding messenger RNA (mRNA). In situ polymerase chain reaction (PCR) (PCR in situ hybridization = PISH) and the new in situ self-sustained sequence replication-based amplification (3SR) method even allow the detection of single copies of DNA or RNA in cytological and histological material. However, there is a number of considerable problems with the in situ PCR methods available today: False positives due to mis-priming of DNA breakdown products contained in several types of cells causing non-specific incorporation of label in direct methods, and re-diffusion artefacts of amplicons into previously negative cells have been observed. To avoid these problems, super-sensitive ISH procedures can be used, and it is well known that the sensitivity and outcome of these methods partially depend on the detection system used.

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