Computational design of mRNA vaccines.

mRNA technology has emerged as a successful vaccine platform that offered a swift response to the COVID-19 pandemic. Accumulating evidence shows that vaccine efficacy, thermostability, and other important properties, are largely impacted by intrinsic properties of the mRNA molecule, such as RNA sequence and structure, both of which can be optimized. Designing mRNA sequence for vaccines presents a combinatorial problem due to an extremely large selection space. For instance, due to the degeneracy of the genetic code, there are over 10632 possible mRNA sequences that could encode the spike protein, the COVID-19 vaccines' target. Moreover, designing different elements of the mRNA sequence simultaneously against multiple objectives such as translational efficiency, reduced reactogenicity, and improved stability requires an efficient and sophisticated optimization strategy. Recently, there has been a growing interest in utilizing computational tools to redesign mRNA sequences to improve vaccine characteristics and expedite discovery timelines. In this review, we explore important biophysical features of mRNA to be considered for vaccine design and discuss how computational approaches can be applied to rapidly design mRNA sequences with desirable characteristics.

tRNA therapeutics for genetic diseases.

Transfer RNAs (tRNAs) have a crucial role in protein synthesis, and in recent years, their therapeutic potential for the treatment of genetic diseases - primarily those associated with a mutation altering mRNA translation - has gained significant attention. Engineering tRNAs to readthrough nonsense mutation-associated premature termination of mRNA translation can restore protein synthesis and function. In addition, supplementation of natural tRNAs can counteract effects of missense mutations in proteins crucial for tRNA biogenesis and function in translation. This Review will present advances in the development of tRNA therapeutics with high activity and safety in vivo and discuss different formulation approaches for single or chronic treatment modalities. The field of tRNA therapeutics is still in its early stages, and a series of challenges related to tRNA efficacy and stability in vivo, delivery systems with tissue-specific tropism, and safe and efficient manufacturing need to be addressed.

Citrullination modulates antigen processing and presentation by revealing cryptic epitopes in rheumatoid arthritis.

Cryptic peptides, hidden from the immune system under physiologic conditions, are revealed by changes to MHC class II processing and hypothesized to drive the loss of immune tolerance to self-antigens in autoimmunity. Rheumatoid arthritis (RA) is an autoimmune disease characterized by immune responses to citrullinated self-antigens, in which arginine residues are converted to citrullines. Here, we investigate the hypothesis that citrullination exposes cryptic peptides by modifying protein structure and proteolytic cleavage. We show that citrullination alters processing and presentation of autoantigens, resulting in the generation of a unique citrullination-dependent repertoire composed primarily of native sequences. This repertoire stimulates T cells from RA patients with anti-citrullinated protein antibodies more robustly than controls. The generation of this unique repertoire is achieved through altered protease cleavage and protein destabilization, rather than direct presentation of citrulline-containing epitopes, suggesting a novel paradigm for the role of protein citrullination in the breach of immune tolerance in RA.

Oligodendrocyte differentiation alters tRNA modifications and codon optimality-mediated mRNA decay.

Oligodendrocytes are specialized cells that confer neuronal myelination in the central nervous system. Leukodystrophies associated with oligodendrocyte deficits and hypomyelination are known to result when a number of tRNA metabolism genes are mutated. Thus, for unknown reasons, oligodendrocytes may be hypersensitive to perturbations in tRNA biology. In this study, we survey the tRNA transcriptome in the murine oligodendrocyte cell lineage and find that specific tRNAs are hypomodified in oligodendrocytes within or near the anticodon compared to oligodendrocyte progenitor cells (OPCs). This hypomodified state may be the result of differential expression of key modification enzymes during oligodendrocyte differentiation. Moreover, we observe a concomitant relationship between tRNA hypomodification and tRNA decoding potential; observing oligodendrocyte specific alterations in codon optimality-mediated mRNA decay and ribosome transit. Our results reveal that oligodendrocytes naturally maintain a delicate, hypersensitized tRNA/mRNA axis. We suggest this axis is a potential mediator of pathology in leukodystrophies and white matter disease when further insult to tRNA metabolism is introduced.

Development of a Tethered mRNA Amplifier to increase protein expression.

Herein, we present a novel method to specifically increase a messenger RNA's (mRNA) expression at the post-transcriptional level. This is accomplished using what we term a "Tethered mRNA Amplifier." The Tethered mRNA Amplifier specifically binds an mRNA's 3' untranslated region and enhances its stability/translation, often doubling protein output. We test this approach on several transcripts associated with haploinsufficiency disorders and increase their steady-state expression in cell culture. We suggest this approach may be a tenable therapeutic modality with precise activity and broad-spectrum application.

Codon optimality-mediated mRNA degradation: Linking translational elongation to mRNA stability.

Messenger RNA (mRNA) translation by the ribosome represents the final step of a complicated molecular dance from DNA to protein. Although classically considered a decipherer that translates a 64-word genetic code into a proteome of astonishing complexity, the ribosome can also shape the transcriptome by controlling mRNA stability. Recent work has discovered that the ribosome is an arbiter of the general mRNA degradation pathway, wherein the ribosome transit rate serves as a major determinant of transcript half-lives. Specifically, members of the degradation complex sense ribosome translocation rates as a function of ribosome elongation rates. Central to this notion is the concept of codon optimality: although all codons impact translation rates, some are deciphered quickly, whereas others cause ribosome hesitation as a consequence of relative cognate tRNA concentration. These transient pauses induce a unique ribosome conformational state that is probed by the deadenylase complex, thereby inducing an orchestrated set of events that enhance both poly(A) shortening and cap removal. Together, these data imply that the coding region of an mRNA not only encodes for protein content but also impacts protein levels through determining the transcript's fate.

Suppression of premature transcription termination leads to reduced mRNA isoform diversity and neurodegeneration.

Tight regulation of mRNA isoform expression is essential for neuronal development, maintenance, and function; however, the repertoire of proteins that govern isoform composition and abundance remains incomplete. Here, we show that the RNA kinase CLP1 regulates mRNA isoform expression through suppression of proximal cleavage and polyadenylation. We found that human stem-cell-derived motor neurons without CLP1 or with the disease-associated CLP1 p.R140H variant had distinct patterns of RNA-polymerase-II-associated cleavage and polyadenylation complex proteins that correlated with polyadenylation site usage. These changes resulted in imbalanced mRNA isoform expression of long genes important for neuronal function that were recapitulated in vivo. Strikingly, we observed the same pattern of reduced mRNA isoform diversity in 3' end sequencing data from brain tissues of patients with neurodegenerative disease. Together, our results identify a previously uncharacterized role for CLP1 in mRNA 3' end formation and reveal an mRNA misprocessing signature in neurodegeneration that may suggest a common mechanism of disease.

Roles of mRNA poly(A) tails in regulation of eukaryotic gene expression.

In eukaryotes, poly(A) tails are present on almost every mRNA. Early experiments led to the hypothesis that poly(A) tails and the cytoplasmic polyadenylate-binding protein (PABPC) promote translation and prevent mRNA degradation, but the details remained unclear. More recent data suggest that the role of poly(A) tails is much more complex: poly(A)-binding protein can stimulate poly(A) tail removal (deadenylation) and the poly(A) tails of stable, highly translated mRNAs at steady state are much shorter than expected. Furthermore, the rate of translation elongation affects deadenylation. Consequently, the interplay between poly(A) tails, PABPC, translation and mRNA decay has a major role in gene regulation. In this Review, we discuss recent work that is revolutionizing our understanding of the roles of poly(A) tails in the cytoplasm. Specifically, we discuss the roles of poly(A) tails in translation and control of mRNA stability and how poly(A) tails are removed by exonucleases (deadenylases), including CCR4-NOT and PAN2-PAN3. We also discuss how deadenylation rate is determined, the integration of deadenylation with other cellular processes and the function of PABPC. We conclude with an outlook for the future of research in this field.

Quantitative tRNA-sequencing uncovers metazoan tissue-specific tRNA regulation.

Transfer RNAs (tRNA) are quintessential in deciphering the genetic code; disseminating nucleic acid triplets into correct amino acid identity. While this decoding function is clear, an emerging theme is that tRNA abundance and functionality can powerfully impact protein production rate, folding, activity, and messenger RNA stability. Importantly, however, the expression pattern of tRNAs is obliquely known. Here we present Quantitative Mature tRNA sequencing (QuantM-tRNA seq), a technique to monitor tRNA abundance and sequence variants secondary to RNA modifications. With QuantM-tRNA seq, we assess the tRNA transcriptome in mammalian tissues. We observe dramatic distinctions in isodecoder expression and known tRNA modifications between tissues. Remarkably, despite dramatic changes in tRNA isodecoder gene expression, the overall anticodon pool of each tRNA family is similar across tissues. These findings suggest that while anticodon pools appear to be buffered via an unknown mechanism, underlying transcriptomic and epitranscriptomic differences suggest a more complex tRNA regulatory landscape.

The Ccr4-Not complex monitors the translating ribosome for codon optimality.

Control of messenger RNA (mRNA) decay rate is intimately connected to translation elongation, but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. We used a combination of cryo-electron microscopy, ribosome profiling, and mRNA stability assays to examine the recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site in Saccharomyces cerevisiae This interaction occurred when the ribosome lacked accommodated A-site transfer RNA, indicative of low codon optimality. Loss of the interaction resulted in the inability of the mRNA degradation machinery to sense codon optimality. Our findings elucidate a physical link between the Ccr4-Not complex and the ribosome and provide mechanistic insight into the coupling of decoding efficiency with mRNA stability.

Codon and amino acid content are associated with mRNA stability in mammalian cells.

Messenger RNA (mRNA) degradation plays a critical role in regulating transcript levels in the cell and is a major control point for modulating gene expression. In yeast and other model organisms, codon identity is a powerful determinant of transcript stability, contributing broadly to impact half-lives. General principles governing mRNA stability are poorly understood in mammalian systems. Importantly, however, the degradation machinery is highly conserved, thus it seems logical that mammalian transcript half-lives would also be strongly influenced by coding determinants. Herein we characterize the contribution of coding sequence towards mRNA decay in human and Chinese Hamster Ovary cells. In agreement with previous studies, we observed that synonymous codon usage impacts mRNA stability in mammalian cells. Surprisingly, however, we also observe that the amino acid content of a gene is an additional determinant correlating with transcript stability. The impact of codon and amino acid identity on mRNA decay appears to be associated with underlying tRNA and intracellular amino acid concentrations. Accordingly, genes of similar physiological function appear to coordinate their mRNA stabilities in part through codon and amino acid content. Together, these results raise the possibility that intracellular tRNA and amino acid levels interplay to mediate coupling between translational elongation and mRNA degradation rate in mammals.

Regulation of MicroRNA Machinery and Development by Interspecies S-Nitrosylation.

Bioactive molecules can pass between microbiota and host to influence host cellular functions. However, general principles of interspecies communication have not been discovered. We show here in C. elegans that nitric oxide derived from resident bacteria promotes widespread S-nitrosylation of the host proteome. We further show that microbiota-dependent S-nitrosylation of C. elegans Argonaute protein (ALG-1)-at a site conserved and S-nitrosylated in mammalian Argonaute 2 (AGO2)-alters its function in controlling gene expression via microRNAs. By selectively eliminating nitric oxide generation by the microbiota or S-nitrosylation in ALG-1, we reveal unforeseen effects on host development. Thus, the microbiota can shape the post-translational landscape of the host proteome to regulate microRNA activity, gene expression, and host development. Our findings suggest a general mechanism by which the microbiota may control host cellular functions, as well as a new role for gasotransmitters.

Publisher Correction: Structural and molecular mechanisms for the control of eukaryotic 5'-3' mRNA decay.

The original and corrected figures are shown in the accompanying Publisher Correction.

Structural and molecular mechanisms for the control of eukaryotic 5'-3' mRNA decay.

5'-3' RNA decay pathways are critical for quality control and regulation of gene expression. Structural and biochemical studies have provided insights into the key nucleases that carry out deadenylation, decapping, and exonucleolysis during 5'-3' decay, but detailed understanding of how these activities are coordinated is only beginning to emerge. Here we review recent mechanistic insights into the control of 5'-3' RNA decay, including coupling between translation and decay, coordination between the complexes and activities that process 5' and 3' RNA termini, conformational control of enzymatic activity, liquid phase separation, and RNA modifications.

Attenuated Codon Optimality Contributes to Neural-Specific mRNA Decay in Drosophila.

Tissue-specific mRNA stability is important for cell fate and physiology, but the mechanisms involved are not fully understood. We found that zygotic mRNA stability in Drosophila correlates with codon content: optimal codons are enriched in stable transcripts associated with metabolic functions like translation, while non-optimal codons are enriched in unstable transcripts, including those associated with neural development. Bioinformatic analyses and reporter assays revealed that similar codons stabilize or destabilize mRNAs in the nervous system and other tissues, but the link between codon content and stability is attenuated in the nervous system. We confirmed that optimal codons are decoded by abundant tRNAs while non-optimal codons are decoded by less abundant tRNAs in embryos and in the nervous system. We conclude that codon optimality is a general determinant of zygotic mRNA stability, and attenuation of codon optimality allows trans-acting factors to exert greater influence over mRNA decay in the nervous system.

Translation elongation and mRNA stability are coupled through the ribosomal A-site.

Messenger RNA (mRNA) degradation plays a critical role in regulating transcript levels in eukaryotic cells. Previous work by us and others has shown that codon identity exerts a powerful influence on mRNA stability. In Saccharomyces cerevisiae, studies using a handful of reporter mRNAs show that optimal codons increase translation elongation rate, which in turn increases mRNA stability. However, a direct relationship between elongation rate and mRNA stability has not been established across the entire yeast transcriptome. In addition, there is evidence from work in higher eukaryotes that amino acid identity influences mRNA stability, raising the question as to whether the impact of translation elongation on mRNA decay is at the level of tRNA decoding, amino acid incorporation, or some combination of each. To address these questions, we performed ribosome profiling of wild-type yeast. In good agreement with other studies, our data showed faster codon-specific elongation over optimal codons and faster transcript-level elongation correlating with transcript optimality. At both the codon-level and transcript-level, faster elongation correlated with increased mRNA stability. These findings were reinforced by showing increased translation efficiency and kinetics for a panel of 11 HIS3 reporter mRNAs of increasing codon optimality. While we did observe that elongation measured by ribosome profiling is composed of both amino acid identity and synonymous codon effects, further analyses of these data establish that A-site tRNA decoding rather than other steps of translation elongation is driving mRNA decay in yeast.

mRNA Deadenylation Is Coupled to Translation Rates by the Differential Activities of Ccr4-Not Nucleases.

Translation and decay of eukaryotic mRNAs is controlled by shortening of the poly(A) tail and release of the poly(A)-binding protein Pab1/PABP. The Ccr4-Not complex contains two exonucleases-Ccr4 and Caf1/Pop2-that mediate mRNA deadenylation. Here, using a fully reconstituted biochemical system with proteins from the fission yeast Schizosaccharomyces pombe, we show that Pab1 interacts with Ccr4-Not, stimulates deadenylation, and differentiates the roles of the nuclease enzymes. Surprisingly, Pab1 release relies on Ccr4 activity. In agreement with this, in vivo experiments in budding yeast show that Ccr4 is a general deadenylase that acts on all mRNAs. In contrast, Caf1 only trims poly(A) not bound by Pab1. As a consequence, Caf1 is a specialized deadenylase required for the selective deadenylation of transcripts with lower rates of translation elongation and reduced Pab1 occupancy. These findings reveal a coupling between the rates of translation and deadenylation that is dependent on Pab1 and Ccr4-Not.

Codon optimality, bias and usage in translation and mRNA decay.

The advent of ribosome profiling and other tools to probe mRNA translation has revealed that codon bias - the uneven use of synonymous codons in the transcriptome - serves as a secondary genetic code: a code that guides the efficiency of protein production, the fidelity of translation and the metabolism of mRNAs. Recent advancements in our understanding of mRNA decay have revealed a tight coupling between ribosome dynamics and the stability of mRNA transcripts; this coupling integrates codon bias into the concept of codon optimality, or the effects that specific codons and tRNA concentrations have on the efficiency and fidelity of the translation machinery. In this Review, we first discuss the evidence for codon-dependent effects on translation, beginning with the basic mechanisms through which translation perturbation can affect translation efficiency, protein folding and transcript stability. We then discuss how codon effects are leveraged by the cell to tailor the proteome to maintain homeostasis, execute specific gene expression programmes of growth or differentiation and optimize the efficiency of protein production.

Short poly(A) tails are a conserved feature of highly expressed genes.

Poly(A) tails are important elements in mRNA translation and stability, although recent genome-wide studies have concluded that poly(A) tail length is generally not associated with translational efficiency in nonembryonic cells. To investigate whether poly(A) tail size might be coupled to gene expression in an intact organism, we used an adapted TAIL-seq protocol to measure poly(A) tails in Caenorhabditis elegans. Surprisingly, we found that well-expressed transcripts contain relatively short, well-defined tails. This attribute appears to be dependent on translational efficiency, as transcripts enriched for optimal codons and ribosome association had the shortest tail sizes, whereas noncoding RNAs retained long tails. Across eukaryotes, short tails were a feature of abundant and well-translated mRNAs. This seems to contradict the dogma that deadenylation induces translational inhibition and mRNA decay and suggests that well-expressed mRNAs accumulate with pruned tails that accommodate a minimal number of poly(A)-binding proteins, which may be ideal for protective and translational functions.