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Science Advances Jun 2023Quantifying aging rate is important for evaluating age-associated decline and mortality. A blood single-cell RNA sequencing dataset for seven supercentenarians (SCs) was...
Quantifying aging rate is important for evaluating age-associated decline and mortality. A blood single-cell RNA sequencing dataset for seven supercentenarians (SCs) was recently generated. Here, we generate a reference 28-sample aging cohort to compute a single-cell level aging clock and to determine the biological age of SCs. Our clock model placed the SCs at a blood biological age to between 80.43 and 102.67 years. Compared to the model-expected aging trajectory, SCs display increased naive CD8 T cells, decreased cytotoxic CD8 T cells, memory CD4 T cells, and megakaryocytes. As the most prominent molecular hallmarks at the single-cell level, SCs contain more cells and cell types with high ribosome level, which is associated with and, according to Bayesian network inference, contributes to a low inflammation state and slow aging of SCs. Inhibiting ribosomal activity or translation in monocytes validates such translation against inflammation balance revealed by our single-cell aging clock.
Topics: Humans; Aged, 80 and over; Longevity; CD8-Positive T-Lymphocytes; Leukocytes, Mononuclear; Bayes Theorem; Single-Cell Gene Expression Analysis; Aging; Cellular Senescence; Inflammation; Ribosomes
PubMed: 37379396
DOI: 10.1126/sciadv.abq7599 -
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 -
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 -
PloS One 2023The continuing emergence of SARS-CoV-2 variants has highlighted the need to identify additional points for viral inhibition. Ribosome inactivating proteins (RIPs), such...
The continuing emergence of SARS-CoV-2 variants has highlighted the need to identify additional points for viral inhibition. Ribosome inactivating proteins (RIPs), such as MAP30 and Momordin which are derived from bitter melon (Momordica charantia), have been found to inhibit a broad range of viruses. MAP30 has been shown to potently inhibit HIV-1 with minimal cytotoxicity. Here we show that MAP30 and Momordin potently inhibit SARS-CoV-2 replication in A549 human lung cells (IC50 ~ 0.2 μM) with little concomitant cytotoxicity (CC50 ~ 2 μM). Both viral inhibition and cytotoxicity remain unaltered by appending a C-terminal Tat cell-penetration peptide to either protein. Mutation of tyrosine 70, a key residue in the active site of MAP30, to alanine completely abrogates both viral inhibition and cytotoxicity, indicating the involvement of its RNA N-glycosylase activity. Mutation of lysine 171 and lysine 215, residues corresponding to those in Ricin which when mutated prevented ribosome binding and inactivation, to alanine in MAP30 decreased cytotoxicity (CC50 ~ 10 μM) but also the viral inhibition (IC50 ~ 1 μM). Unlike with HIV-1, neither Dexamethasone nor Indomethacin exhibited synergy with MAP30 in the inhibition of SARS-CoV-2. From a structural comparison of the two proteins, one can explain their similar activities despite differences in both their active-sites and ribosome-binding regions. We also note points on the viral genome for potential inhibition by these proteins.
Topics: Humans; COVID-19; Lysine; SARS-CoV-2; Alanine; HIV Seropositivity; HIV-1; Momordica charantia; Ribosome Inactivating Proteins; Ribosomes; COVID-19 Drug Treatment
PubMed: 37384752
DOI: 10.1371/journal.pone.0286370 -
Cell Jul 2023Readthrough into the 3' untranslated region (3' UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but...
Readthrough into the 3' untranslated region (3' UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but the underlying mechanisms remain unknown. Here, we show in Caenorhabditis elegans and mammalian cells that readthrough proteins are targeted by a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome-collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions (CTEs) are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay initiated by GCN1 and CCR4/NOT limits the accumulation of readthrough products. Unexpectedly, selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes collide at nonoptimal codons, enriched in 3' UTRs, transmembrane proteins, and collagens. GCN1 dysfunction increasingly perturbs these protein classes during aging, resulting in mRNA and proteome imbalance. Our results define GCN1 as a key factor acting during translation in maintaining protein homeostasis.
Topics: Animals; Protein Biosynthesis; Ribosomes; Proteins; RNA, Messenger; Codon, Terminator; Mammals
PubMed: 37339632
DOI: 10.1016/j.cell.2023.05.035 -
Cell Nov 2023Mammalian oocytes are filled with poorly understood structures called cytoplasmic lattices. First discovered in the 1960s and speculated to correspond to mammalian yolk,...
Mammalian oocytes are filled with poorly understood structures called cytoplasmic lattices. First discovered in the 1960s and speculated to correspond to mammalian yolk, ribosomal arrays, or intermediate filaments, their function has remained enigmatic to date. Here, we show that cytoplasmic lattices are sites where oocytes store essential proteins for early embryonic development. Using super-resolution light microscopy and cryoelectron tomography, we show that cytoplasmic lattices are composed of filaments with a high surface area, which contain PADI6 and subcortical maternal complex proteins. The lattices associate with many proteins critical for embryonic development, including proteins that control epigenetic reprogramming of the preimplantation embryo. Loss of cytoplasmic lattices by knocking out PADI6 or the subcortical maternal complex prevents the accumulation of these proteins and results in early embryonic arrest. Our work suggests that cytoplasmic lattices enrich maternally provided proteins to prevent their premature degradation and cellular activity, thereby enabling early mammalian development.
Topics: Pregnancy; Animals; Female; Oocytes; Proteins; Embryo, Mammalian; Cytoskeleton; Ribosomes; Embryonic Development; Mammals
PubMed: 37922900
DOI: 10.1016/j.cell.2023.10.003 -
Nature Jan 2024In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2...
In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IVT mRNAs are transfected into target cells, where they are translated into recombinant protein, and the biological activity or immunogenicity of the encoded protein exerts an intended therapeutic effect. Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to decrease their innate immunogenicity, but their effects on mRNA translation fidelity have not been fully explored. Here we demonstrate that incorporation of N-methylpseudouridine into mRNA results in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurs after vaccination. The +1 ribosome frameshifting observed is probably a consequence of N-methylpseudouridine-induced ribosome stalling during IVT mRNA translation, with frameshifting occurring at ribosome slippery sequences. However, we demonstrate that synonymous targeting of such slippery sequences provides an effective strategy to reduce the production of frameshifted products. Overall, these data increase our understanding of how modified ribonucleotides affect the fidelity of mRNA translation, and although there are no adverse outcomes reported from mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, these data highlight potential off-target effects for future mRNA-based therapeutics and demonstrate the requirement for sequence optimization.
Topics: Animals; Humans; Mice; BNT162 Vaccine; Frameshifting, Ribosomal; RNA, Messenger; Pseudouridine; Ribosomes; Protein Biosynthesis
PubMed: 38057663
DOI: 10.1038/s41586-023-06800-3 -
Nature Jun 2023Translation regulation is critical for early mammalian embryonic development. However, previous studies had been restricted to bulk measurements, precluding precise...
Translation regulation is critical for early mammalian embryonic development. However, previous studies had been restricted to bulk measurements, precluding precise determination of translation regulation including allele-specific analyses. Here, to address this challenge, we developed a novel microfluidic isotachophoresis (ITP) approach, named RIBOsome profiling via ITP (Ribo-ITP), and characterized translation in single oocytes and embryos during early mouse development. We identified differential translation efficiency as a key mechanism regulating genes involved in centrosome organization and N-methyladenosine modification of RNAs. Our high-coverage measurements enabled, to our knowledge, the first analysis of allele-specific ribosome engagement in early development. These led to the discovery of stage-specific differential engagement of zygotic RNAs with ribosomes and reduced translation efficiency of transcripts exhibiting allele-biased expression. By integrating our measurements with proteomics data, we discovered that ribosome occupancy in germinal vesicle-stage oocytes is the predominant determinant of protein abundance in the zygote. The Ribo-ITP approach will enable numerous applications by providing high-coverage and high-resolution ribosome occupancy measurements from ultra-low input samples including single cells.
Topics: Animals; Mice; Protein Biosynthesis; Proteomics; Ribosomes; RNA, Messenger; Single-Cell Analysis; Embryonic Development; Alleles; Microfluidic Analytical Techniques; Oocytes; Isotachophoresis; Ribosome Profiling; Centrosome; Zygote
PubMed: 37344592
DOI: 10.1038/s41586-023-06228-9 -
Molecular Cell Nov 2023tRNA function is based on unique structures that enable mRNA decoding using anticodon trinucleotides. These structures interact with specific aminoacyl-tRNA synthetases... (Review)
Review
tRNA function is based on unique structures that enable mRNA decoding using anticodon trinucleotides. These structures interact with specific aminoacyl-tRNA synthetases and ribosomes using 3D shape and sequence signatures. Beyond translation, tRNAs serve as versatile signaling molecules interacting with other RNAs and proteins. Through evolutionary processes, tRNA fragmentation emerges as not merely random degradation but an act of recreation, generating specific shorter molecules called tRNA-derived small RNAs (tsRNAs). These tsRNAs exploit their linear sequences and newly arranged 3D structures for unexpected biological functions, epitomizing the tRNA "renovatio" (from Latin, meaning renewal, renovation, and rebirth). Emerging methods to uncover full tRNA/tsRNA sequences and modifications, combined with techniques to study RNA structures and to integrate AI-powered predictions, will enable comprehensive investigations of tRNA fragmentation products and new interaction potentials in relation to their biological functions. We anticipate that these directions will herald a new era for understanding biological complexity and advancing pharmaceutical engineering.
Topics: RNA, Transfer; Anticodon; Amino Acyl-tRNA Synthetases; Ribosomes; RNA, Messenger
PubMed: 37802077
DOI: 10.1016/j.molcel.2023.09.016 -
Nature Communications Oct 2023Tumor growth requires elevated ribosome biogenesis. Targeting ribosomes is an important strategy for cancer therapy. The ribosome inhibitor, homoharringtonine (HHT), is...
Tumor growth requires elevated ribosome biogenesis. Targeting ribosomes is an important strategy for cancer therapy. The ribosome inhibitor, homoharringtonine (HHT), is used for the clinical treatment of leukemia, yet it is ineffective for the treatment of solid tumors, the reasons for which remain unclear. Here we show that Snail1, a key factor in the regulation of epithelial-to-mesenchymal transition, plays a pivotal role in cellular surveillance response upon ribotoxic stress. Mechanistically, ribotoxic stress activates the JNK-USP36 signaling to stabilize Snail1 in the nucleolus, which facilitates ribosome biogenesis and tumor cell survival. Furthermore, we show that HHT activates the JNK-USP36-Snail1 axis in solid tumor cells, but not in leukemia cells, resulting in solid tumor cell resistance to HHT. Importantly, a combination of HHT with the inhibition of the JNK-USP36-Snail1 axis synergistically inhibits solid tumor growth. Together, this study provides a rationale for targeting the JNK-USP36-Snail1 axis in ribosome inhibition-based solid tumor therapy.
Topics: Humans; Cell Survival; Ribosomes; Cell Nucleolus; Neoplasms; Leukemia; Ubiquitin Thiolesterase
PubMed: 37833415
DOI: 10.1038/s41467-023-42257-8