mRNA structure regulates protein expression through changes in functional half-life

Mauger, David; Cabral, B. Joseph; Presnyak, Vladimir; Su, Stephen; Reid, David W.; Goodman, Brooke; Link, Kristian H.; Khatwani, Nikhil; Reynders, John; Moore, Melissa J.; McFadyen, Iain

doi:10.1101/549022

Cited by 53 publications

(71 citation statements)

References 47 publications

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“…For all four challenges, the sequences with the lowest AUP values were designed by Eterna participants ( Figure 3C). (35). The minimal AUP value from those sequences was 0.381, was similar to the value obtained from randomly-sampled codons, suggesting that explicit optimization of AUP is necessary for applications seeking stability against hydrolysis.…”

Section: Introductionsupporting

confidence: 61%

“…The choice of eGFP+degron allowed for close comparison with designed mRNAs from ref. (35) , which investigated the relationships between mRNA structure, stability and translation using the eGFP+degron, while Nanoluciferase was chosen to complement another easily assayed, commonly used model protein in mammalian systems.…”

Section: Methodsmentioning

confidence: 99%

“…The four systems were a multi-epitope vaccine design (MEV) derived from SARS-CoV-2 spike (S) and nucleocapsid (N) proteins; Nanoluciferase; enhanced green fluorescence protein with an attached degron sequence (eGFP+deg), used by Mauger et al for characterizing mRNA stability and translation; and the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. We compared sequences generated by a variety of methods ( Figure 1D): uniform sampling of codons ("Uniform random codons"); uniform sampling of GC-rich codons only ("GC-rich codons"); vendor-supplied servers from IDT, GENEWIZ, and Twist; and the algorithm LinearDesign (29), which returns a sequence with minimal ΔG(MFE) solution that is balanced with codon usage, as well as sequences from other groups when possible (35). We further developed a stochastic Monte Carlo Tree Search algorithm, RiboTree, to stochastically minimize AUP of model mRNAs.…”

Section: Introductionmentioning

confidence: 99%

“…Sequences designed rationally by participants during Eterna's OpenVaccine challenge result in the lowest AUP values for mRNAs encoding a variety of model proteins used for studying translation and as model vaccines, ranging in length from 144 nucleotides (the Multi-epitope Vaccine) to 855 nucleotides (eGFP+degron(35)). (A) Eterna designs encompass a diverse sequence space when compared to mRNA sequences generated by random sampling, visualized with a principal component analysis.…”

mentioning

confidence: 99%

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Theoretical basis for stabilizing messenger RNA through secondary structure design

Wayment-Steele

Kim

Choe

et al. 2020

Preprint

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RNA hydrolysis presents problems in manufacturing, long-term storage, world-wide delivery, and in vivo stability of messenger RNA (mRNA)-based vaccines and therapeutics. A largely unexplored strategy to reduce mRNA hydrolysis is to redesign RNAs to form double-stranded regions, which are protected from in-line cleavage and enzymatic degradation, while coding for the same proteins. The amount of stabilization that this strategy can deliver and the most effective algorithmic approach to achieve stabilization remain poorly understood. Motivated by the need for stabilized COVID-19 mRNA vaccines, we present simple calculations for estimating RNA stability against hydrolysis, and a model that links the average unpaired probability of an mRNA, or AUP, to its overall rate of hydrolysis. To characterize the stabilization achievable through structure design, we compare optimization of AUP by conventional mRNA design methods to results from the LinearDesign algorithm, a new Monte Carlo tree search algorithm called RiboTree, and crowdsourcing through the OpenVaccine challenge on the Eterna platform. Tests were carried out on mRNAs encoding nanoluciferase, green fluorescent protein, and COVID-19 mRNA vaccine candidates encoding SARS-CoV-2 epitopes, spike receptor binding domain, and full-length spike protein. We find that Eterna and RiboTree significantly lower AUP while maintaining a large diversity of sequence and structure features that correlate with translation, biophysical size, and immunogenicity. Our results suggest that increases in in vitro mRNA half-life by at least two-fold are immediately achievable and that further stability improvements may be enabled with thorough experimental characterization of RNA hydrolysis.

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Section: Introductionsupporting

confidence: 61%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Theoretical basis for stabilizing messenger RNA through secondary structure design

Wayment-Steele

Kim

Choe

et al. 2020

Preprint

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“…Additionally, lower resolution RNA‐secondary structure mapping studies (e.g. using DMS or SHAPE reagents) comparing modified and unmodified mRNAs can provide additional insights information about how the shape of mRNAs is influenced by modifications (Mauger et al, ). Structural studies will prove useful not only for understanding how modifications impact the intrinsic properties of mRNAs to impact their function and stability, but also as researchers are seeking to develop therapeutics targeting m 6 A‐modified transcripts (Cross, ).…”

Section: Quantitative Approaches For Studying Mrna Modification Levelmentioning

confidence: 99%

A molecular‐level perspective on the frequency, distribution, and consequences of messenger RNA modifications

2020

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Cells use chemical modifications to alter the sterics, charge, and conformations of large biomolecules, modulating their biogenesis, function, and stability. Until recently post‐transcriptional RNA modifications were thought to be largely limited to nonprotein coding RNA species. However, this dogma has rapidly transformed with the discovery of a host of modifications in protein coding messenger RNAs (mRNAs). Recent advancements in genome‐wide sequencing technologies have enabled the identification of mRNA modifications as a potential new frontier in gene regulation—leading to the development of the epitranscriptome field. As a result, there has been a flurry of multiple groundbreaking discoveries, including new modifications, nucleoside modifying enzymes (“writers” and “erasers”), and RNA binding proteins that recognize chemical modifications (“readers”). These discoveries opened the door to understanding how post‐transcriptional mRNA modifications can modulate the mRNA lifecycle, and established a link between the epitranscriptome and human health and disease. Despite a rapidly growing recognition of their importance, fundamental questions regarding the identity, prevalence, and functional consequences of mRNA modifications remain to be answered. Here, we highlight quantitative studies that characterize mRNA modification abundance, frequency, and interactions with cellular machinery. As the field progresses, we see a need for the further integration of quantitative and reductionist approaches to complement transcriptome wide studies in order to establish a molecular‐level framework for understanding the consequences of mRNA chemical modifications on biological processes. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Processing > RNA Editing and Modification

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Synthetic in vitro transcribed lncRNAs (SINEUPs) with chemical modifications enhance target mRNA translation

et al. 2020

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Chemically modified mRNAs are extensively studied with a view toward their clinical application. In particular, long noncoding RNAs (lncRNAs) containing SINE elements, which enhance the translation of their target mRNAs (i.e., SINEUPs), have potential as RNA therapies for various diseases, such as haploinsufficiencies. To establish a SINEUP-based system for efficient protein expression, we directly transfected chemically modified in vitro transcribed (mIVT) SINEUP RNAs to examine their effects on target mRNA translation. mIVT SINEUP RNAs enhanced translation of EGFP mRNA and endogenous target Sox9 mRNA in both cultured cells and a cell-free translation system. Our findings reveal the functional role of RNA modifications in SINEUPs and suggest several broad clinical applications of such an RNA regulatory system. Keywords: enhancement of endogenous target mRNA translation; in vitro transcribed RNA; nucleic acid-based therapeutics; RNA modification Abbreviations BD, binding domain; ED, effector domain; EGFP, enhanced green fluorescent protein; FISH, fluorescence in situ hybridization; IVT RNAs, in vitro transcribed RNAs; IVT, in vitro transcribed; lncRNA, long noncoding RNA; m, chemically modified; RBP, RNA-binding protein; RRL, rabbit reticulocyte lysate; SINEUP, lncRNA that contains an inverted SINEB2 repeat element and upregulates the translation of a target mRNA; SINEUP-GFP, SINEUP RNA that contains a binding domain designed to target EGFP mRNA; SINEUP-SCR, SINEUP RNA that contains a scrambled, nonbinding domain instead of the EGFP-binding domain; SOX9, sex-determining region Y-box 9.

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mRNA structure regulates protein expression through changes in functional half-life

Cited by 53 publications

References 47 publications

Theoretical basis for stabilizing messenger RNA through secondary structure design

Theoretical basis for stabilizing messenger RNA through secondary structure design

A molecular‐level perspective on the frequency, distribution, and consequences of messenger RNA modifications

Synthetic in vitro transcribed lncRNAs (SINEUPs) with chemical modifications enhance target mRNA translation

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