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Cell Aug 2023A system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of...
A system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of delivering executable RNA programs to other cells. We developed genetically encoded cellular RNA exporters, inspired by viruses, that efficiently package and secrete cargo RNA molecules from mammalian cells within protective nanoparticles. Exporting and sequencing RNA barcodes enabled non-destructive monitoring of cell population dynamics with clonal resolution. Further, by incorporating fusogens into the nanoparticles, we demonstrated the delivery, expression, and functional activity of exported mRNA in recipient cells. We term these systems COURIER (controlled output and uptake of RNA for interrogation, expression, and regulation). COURIER enables measurement of cell dynamics and establishes a foundation for hybrid cell and gene therapies based on cell-to-cell delivery of RNA.
Topics: Animals; Biological Transport; Mammals; RNA; RNA, Messenger; Viruses; Molecular Typing; Sequence Analysis, RNA; Cytological Techniques; Genetic Techniques
PubMed: 37437570
DOI: 10.1016/j.cell.2023.06.013 -
Science (New York, N.Y.) Jun 2023The precise control of messenger RNA (mRNA) translation is a crucial step in posttranscriptional gene regulation of cellular physiology. However, it remains a challenge...
The precise control of messenger RNA (mRNA) translation is a crucial step in posttranscriptional gene regulation of cellular physiology. However, it remains a challenge to systematically study mRNA translation at the transcriptomic scale with spatial and single-cell resolution. Here, we report the development of ribosome-bound mRNA mapping (RIBOmap), a highly multiplexed three-dimensional in situ profiling method to detect cellular translatome. RIBOmap profiling of 981 genes in HeLa cells revealed cell cycle-dependent translational control and colocalized translation of functional gene modules. We mapped 5413 genes in mouse brain tissues, yielding spatially resolved single-cell translatomic profiles for 119,173 cells and revealing cell type-specific and brain region-specific translational regulation, including translation remodeling during oligodendrocyte maturation. Our method detected widespread patterns of localized translation in neuronal and glial cells in intact brain tissue networks.
Topics: Animals; Humans; Mice; Brain; HeLa Cells; Neuroglia; RNA, Messenger; Gene Expression Regulation; Chromosome Mapping; Protein Biosynthesis; Neurons; Single-Cell Gene Expression Analysis
PubMed: 37384709
DOI: 10.1126/science.add3067 -
Annual Review of Biochemistry Jun 2023Messenger RNA (mRNA) stability and translational efficiency are two crucial aspects of the post-transcriptional process that profoundly impact protein production in a... (Review)
Review
Messenger RNA (mRNA) stability and translational efficiency are two crucial aspects of the post-transcriptional process that profoundly impact protein production in a cell. While it is widely known that ribosomes produce proteins, studies during the past decade have surprisingly revealed that ribosomes also control mRNA stability in a codon-dependent manner, a process referred to as codon optimality. Therefore, codons, the three-nucleotide words read by the ribosome, have a potent effect on mRNA stability and provide cisregulatory information that extends beyond the amino acids they encode. While the codon optimality molecular mechanism is still unclear, the translation elongation rate appears to trigger mRNA decay. Thus, transfer RNAs emerge as potential master gene regulators affecting mRNA stability. Furthermore, while few factors related to codon optimality have been identified in yeast, the orthologous genes in vertebrates do not necessary share the same functions. Here, we discuss codon optimality findings and gene regulation layers related to codon composition in different eukaryotic species.
Topics: Animals; RNA, Messenger; Protein Biosynthesis; Codon; Proteins; RNA Stability; Saccharomyces cerevisiae
PubMed: 37001134
DOI: 10.1146/annurev-biochem-052621-091808 -
Human Vaccines & Immunotherapeutics Dec 2024The research and development of messenger RNA (mRNA) cancer vaccines have gradually overcome numerous challenges through the application of personalized cancer antigens,... (Review)
Review
The research and development of messenger RNA (mRNA) cancer vaccines have gradually overcome numerous challenges through the application of personalized cancer antigens, structural optimization of mRNA, and the development of alternative RNA-based vectors and efficient targeted delivery vectors. Clinical trials are currently underway for various cancer vaccines that encode tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), or immunomodulators. In this paper, we summarize the optimization of mRNA and the emergence of RNA-based expression vectors in cancer vaccines. We begin by reviewing the advancement and utilization of state-of-the-art targeted lipid nanoparticles (LNPs), followed by presenting the primary classifications and clinical applications of mRNA cancer vaccines. Collectively, mRNA vaccines are emerging as a central focus in cancer immunotherapy, offering the potential to address multiple challenges in cancer treatment, either as standalone therapies or in combination with current cancer treatments.
Topics: Humans; Cancer Vaccines; mRNA Vaccines; Neoplasms; Antigens, Neoplasm; RNA, Messenger; RNA; Nanoparticles
PubMed: 38282471
DOI: 10.1080/21645515.2024.2307187 -
Nature Sep 2023Messenger RNA (mRNA) vaccines are being used to combat the spread of COVID-19 (refs. ), but they still exhibit critical limitations caused by mRNA instability and...
Messenger RNA (mRNA) vaccines are being used to combat the spread of COVID-19 (refs. ), but they still exhibit critical limitations caused by mRNA instability and degradation, which are major obstacles for the storage, distribution and efficacy of the vaccine products. Increasing secondary structure lengthens mRNA half-life, which, together with optimal codons, improves protein expression. Therefore, a principled mRNA design algorithm must optimize both structural stability and codon usage. However, owing to synonymous codons, the mRNA design space is prohibitively large-for example, there are around 2.4 × 10 candidate mRNA sequences for the SARS-CoV-2 spike protein. This poses insurmountable computational challenges. Here we provide a simple and unexpected solution using the classical concept of lattice parsing in computational linguistics, where finding the optimal mRNA sequence is analogous to identifying the most likely sentence among similar-sounding alternatives. Our algorithm LinearDesign finds an optimal mRNA design for the spike protein in just 11 minutes, and can concurrently optimize stability and codon usage. LinearDesign substantially improves mRNA half-life and protein expression, and profoundly increases antibody titre by up to 128 times in mice compared to the codon-optimization benchmark on mRNA vaccines for COVID-19 and varicella-zoster virus. This result reveals the great potential of principled mRNA design and enables the exploration of previously unreachable but highly stable and efficient designs. Our work is a timely tool for vaccines and other mRNA-based medicines encoding therapeutic proteins such as monoclonal antibodies and anti-cancer drugs.
Topics: Animals; Humans; Mice; Algorithms; Codon; COVID-19; COVID-19 Vaccines; Half-Life; Herpesvirus 3, Human; mRNA Vaccines; RNA Stability; RNA, Messenger; SARS-CoV-2
PubMed: 37130545
DOI: 10.1038/s41586-023-06127-z -
Molecular Cell Jul 2023Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics...
Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics and microtubule function. When cells sense excess free tubulin, they trigger degradation of the encoding mRNAs, which requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates the decay of tubulin mRNAs is unknown. Here, our biochemical and structural analysis reveals that TTC5 recruits the poorly studied protein SCAPER to the ribosome. SCAPER, in turn, engages the CCR4-NOT deadenylase complex through its CNOT11 subunit to trigger tubulin mRNA decay. SCAPER mutants that cause intellectual disability and retinitis pigmentosa in humans are impaired in CCR4-NOT recruitment, tubulin mRNA degradation, and microtubule-dependent chromosome segregation. Our findings demonstrate how recognition of a nascent polypeptide on the ribosome is physically linked to mRNA decay factors via a relay of protein-protein interactions, providing a paradigm for specificity in cytoplasmic gene regulation.
Topics: Humans; Tubulin; Ribosomes; Microtubules; Homeostasis; RNA, Messenger; RNA Stability; Carrier Proteins; Transcription Factors
PubMed: 37295431
DOI: 10.1016/j.molcel.2023.05.020 -
Cell Mar 2024Biomolecules incur damage during stress conditions, and damage partitioning represents a vital survival strategy for cells. Here, we identified a distinct stress granule...
Biomolecules incur damage during stress conditions, and damage partitioning represents a vital survival strategy for cells. Here, we identified a distinct stress granule (SG), marked by dsRNA helicase DHX9, which compartmentalizes ultraviolet (UV)-induced RNA, but not DNA, damage. Our FANCI technology revealed that DHX9 SGs are enriched in damaged intron RNA, in contrast to classical SGs that are composed of mature mRNA. UV exposure causes RNA crosslinking damage, impedes intron splicing and decay, and triggers DHX9 SGs within daughter cells. DHX9 SGs promote cell survival and induce dsRNA-related immune response and translation shutdown, differentiating them from classical SGs that assemble downstream of translation arrest. DHX9 modulates dsRNA abundance in the DHX9 SGs and promotes cell viability. Autophagy receptor p62 is activated and important for DHX9 SG disassembly. Our findings establish non-canonical DHX9 SGs as a dedicated non-membrane-bound cytoplasmic compartment that safeguards daughter cells from parental RNA damage.
Topics: Cytoplasm; RNA; RNA, Messenger; Stress Granules; Stress, Physiological; Humans; HeLa Cells
PubMed: 38503283
DOI: 10.1016/j.cell.2024.02.028 -
Annual Review of Biochemistry Jun 2023Formation of the 3' end of a eukaryotic mRNA is a key step in the production of a mature transcript. This process is mediated by a number of protein factors that cleave... (Review)
Review
Formation of the 3' end of a eukaryotic mRNA is a key step in the production of a mature transcript. This process is mediated by a number of protein factors that cleave the pre-mRNA, add a poly(A) tail, and regulate transcription by protein dephosphorylation. Cleavage and polyadenylation specificity factor (CPSF) in humans, or cleavage and polyadenylation factor (CPF) in yeast, coordinates these enzymatic activities with each other, with RNA recognition, and with transcription. The site of pre-mRNA cleavage can strongly influence the translation, stability, and localization of the mRNA. Hence, cleavage site selection is highly regulated. The length of the poly(A) tail is also controlled to ensure that every transcript has a similar tail when it is exported from the nucleus. In this review, we summarize new mechanistic insights into mRNA 3'-end processing obtained through structural studies and biochemical reconstitution and outline outstanding questions in the field.
Topics: Humans; RNA, Messenger; RNA Precursors; mRNA Cleavage and Polyadenylation Factors; Saccharomyces cerevisiae; Gene Expression
PubMed: 37001138
DOI: 10.1146/annurev-biochem-052521-012445 -
Proceedings of the National Academy of... Mar 2024Lipid nanoparticles (LNPs) have recently emerged as a powerful and versatile clinically approved platform for nucleic acid delivery, specifically for mRNA vaccines. A... (Review)
Review
Lipid nanoparticles (LNPs) have recently emerged as a powerful and versatile clinically approved platform for nucleic acid delivery, specifically for mRNA vaccines. A major bottleneck in the field is the release of mRNA-LNPs from the endosomal pathways into the cytosol of cells where they can execute their encoded functions. The data regarding the mechanism of these endosomal escape processes are limited and contradicting. Despite extensive research, there is no consensus regarding the compartment of escape, the cause of the inefficient escape and are currently lacking a robust method to detect the escape. Here, we review the currently known mechanisms of endosomal escape and the available methods to study this process. We critically discuss the limitations and challenges of these methods and the possibilities to overcome these challenges. We propose that the development of currently lacking robust, quantitative high-throughput techniques to study endosomal escape is timely and essential. A better understanding of this process will enable better RNA-LNP designs with improved efficiency to unlock new therapeutic modalities.
Topics: Endosomes; Consensus; Cytosol; RNA; RNA, Messenger
PubMed: 38437552
DOI: 10.1073/pnas.2307800120 -
Molecular Cell Dec 2023The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined...
The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined mRNA enrichment in TIS granules (TGs) and the rough endoplasmic reticulum (ER) through particle sorting and isolated cytosolic mRNAs by digitonin extraction. When focusing on genes that encode non-membrane proteins, we observed that 52% have transcripts enriched in specific compartments. Compartment enrichment correlates with a combinatorial code based on mRNA length, exon length, and 3' UTR-bound RNA-binding proteins. Compartment-biased mRNAs differ in the functional classes of their encoded proteins: TG-enriched mRNAs encode low-abundance proteins with strong enrichment of transcription factors, whereas ER-enriched mRNAs encode large and highly expressed proteins. Compartment localization is an important determinant of mRNA and protein abundance, which is supported by reporter experiments showing that redirecting cytosolic mRNAs to the ER increases their protein expression. In summary, the cytoplasm is functionally compartmentalized by local translation environments.
Topics: Endoplasmic Reticulum; Proteins; Cytosol; RNA, Messenger; Protein Transport; Protein Biosynthesis
PubMed: 38134885
DOI: 10.1016/j.molcel.2023.11.025