Gene- and Species-Specific Hox mRNA Translation by Ribosome Expansion Segments

Leppek, Kathrin; Fujii, Kotaro; Quade, Nick; Susanto, Teodorus Theo; Boehringer, Daniel; Lenarčič, Tea; Xue, Shifeng; Genuth, Naomi R.; Ban, Nenad; Barna, Maria

doi:10.1016/j.molcel.2020.10.023

Cited by 50 publications

(92 citation statements)

References 85 publications

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“…As examples of cellular sequences, we included short 5’ UTRs of cellular mRNAs corresponding to highly abundant proteins that have a high translation rate such as ribosomal proteins (RPs) ( RPS25 , RPL31 , RPL38 ), or structural components such as tubulin beta-2B chain ( Tubb2b ) and actin ( ActB) , as well as human collagen, type I, alpha 2 ( hCOL1A2 ) 19 . We further included regulatory 5’ UTR elements such as the 5’ terminal oligopyrimidine (TOP) motif from RPL18 , which promotes translational activation downstream of mTOR 20 , 21 , as well as the Hoxa9 P4 RNA stem-loop which functions as a translation enhancer 22 . 5’ UTRs previously identified in translation efficiency screens such as complement factor 3 ( C3 ), cytochrome P450 2E1 ( CYP2E1 ), and Apolipoprotein A-II ( APOA2 ) 12 , as well as plant rubisco components ( RBCS3B , RBCS1A ) 23 , were also included.…”

Section: Resultsmentioning

confidence: 99%

“…We decided to use Nluc because its short ORF of 621 nt allowed for synthesis and pooled amplification of full-length DNA templates with UTRs of up to 600 nt attached at each end. Employing Nluc further enabled precise quantitative readout for comparing translation efficiencies in follow-up experiments on individual mRNAs through ratiometric measurements including a firefly luciferase (Fluc) mRNA spike-in control in transfection experiments integrated with luciferase luminescence assays 22 . Additional mRNAs encoding for enhanced green fluorescent protein (eGFP) and a shorter candidate multi-epitope vaccine (MEV) 7 were included as controls in some experiments, further discussed below.…”

Section: Resultsmentioning

confidence: 99%

“…1F , Table S2 ). Additionally, we included larger truncations of combined deletions of adjacent stem-loops and introduced the Hoxa9 P4 stem-loop, a 35 nt element that recruits 40S ribosomal subunits to enhance translation 22 , in lieu of the uORF ( Fig. 1F – G , Table S2 ).…”

Section: Resultsmentioning

confidence: 99%

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Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

Leppek

Byeon

Kladwang

et al. 2021

Preprint

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Therapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop a new RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that superfolder mRNAs can be designed to improve both stability and expression that are further enhanced through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

Leppek

Byeon

Kladwang

et al. 2021

Preprint

Self Cite

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“…The Nanoluciferase sequence was taken from ref. (48). The choice of eGFP+degron allowed for close comparison with designed mRNAs from ref.…”

Section: Discussionmentioning

confidence: 99%

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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“…The IRES activity is modulated during development (Ye et al, 1997). More recently, the presence of other IRES elements in the 5'UTR of subsets of mice HoxA mRNAs (Hoxa3,Hoxa4,Hoxa5,Hoxa9 and Hoxa11) have been demonstrated (Leppek et al, 2020;Xue et al, 2015). Some of these IRESes require the presence of the ribosomal protein RpL38 in the ribosome to efficiently initiate translation, thereby explaining the tissue patterning defective phenotype observed with RpL38 knockout mouse (Kondrashov et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Translation Inhibitory Elements from Hox a3 and a11 mRNAs use uORFs for translation inhibition

Martin

Schaeffer

Eriani

et al. 2021

Preprint

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During embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5’UTR. First, an Internal Ribosome Entry Site (IRES) enables cap-independent translation. The second regulon is a Translation Inhibitory Element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of Hox a3 and a11 TIE elements. Both TIEs possess an upstream Open Reading Frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In TIE a3, we identify a uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of TIE a11 is different, it also contains a uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a ‘start-stop’. The a11 ‘start-stop’ sequence is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hox a11 main ORF.

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Gene- and Species-Specific Hox mRNA Translation by Ribosome Expansion Segments

Cited by 50 publications

References 85 publications

Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

Theoretical basis for stabilizing messenger RNA through secondary structure design

Translation Inhibitory Elements from Hox a3 and a11 mRNAs use uORFs for translation inhibition

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