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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 -
Science Advances Aug 2023Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like protein covalently conjugated with intracellular proteins through ufmylation, similar to ubiquitylation. Ufmylation...
Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like protein covalently conjugated with intracellular proteins through ufmylation, similar to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control (RQC) at the ER (ER-RQC), and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here, we provide insights into the mechanism of the UFM1 E3 complex in not only ufmylation but also ER-RQC. The E3 complex consisting of UFL1 and UFBP1 interacted with UFC1, UFM1 E2, and, subsequently, CDK5RAP3, an adaptor for ufmylation of ribosomal subunit RPL26. Upon disome formation, the E3 complex associated with ufmylated RPL26 on the 60 subunit through the UFM1-interacting region of UFBP1. Loss of E3 components or disruption of the interaction between UFBP1 and ufmylated RPL26 attenuated ER-RQC. These results provide insights into not only the molecular basis of the ufmylation but also its role in proteostasis.
Topics: Endoplasmic Reticulum-Associated Degradation; Ribosomes; Ubiquitin-Protein Ligases; Ubiquitination; Ubiquitins; Humans; HEK293 Cells
PubMed: 37595036
DOI: 10.1126/sciadv.adh3635 -
Journal of Hematology & Oncology Feb 2024While liver cancer stem cells (CSCs) play a crucial role in hepatocellular carcinoma (HCC) initiation, progression, recurrence, and treatment resistance, the mechanism...
BACKGROUND
While liver cancer stem cells (CSCs) play a crucial role in hepatocellular carcinoma (HCC) initiation, progression, recurrence, and treatment resistance, the mechanism underlying liver CSC self-renewal remains elusive. We aim to characterize the role of Methyltransferase 16 (METTL16), a recently identified RNA N-methyladenosine (mA) methyltransferase, in HCC development/maintenance, CSC stemness, as well as normal hepatogenesis.
METHODS
Liver-specific Mettl16 conditional KO (cKO) mice were generated to assess its role in HCC pathogenesis and normal hepatogenesis. Hydrodynamic tail-vein injection (HDTVi)-induced de novo hepatocarcinogenesis and xenograft models were utilized to determine the role of METTL16 in HCC initiation and progression. A limiting dilution assay was utilized to evaluate CSC frequency. Functionally essential targets were revealed via integrative analysis of multi-omics data, including RNA-seq, RNA immunoprecipitation (RIP)-seq, and ribosome profiling.
RESULTS
METTL16 is highly expressed in liver CSCs and its depletion dramatically decreased CSC frequency in vitro and in vivo. Mettl16 KO significantly attenuated HCC initiation and progression, yet only slightly influenced normal hepatogenesis. Mechanistic studies, including high-throughput sequencing, unveiled METTL16 as a key regulator of ribosomal RNA (rRNA) maturation and mRNA translation and identified eukaryotic translation initiation factor 3 subunit a (eIF3a) transcript as a bona-fide target of METTL16 in HCC. In addition, the functionally essential regions of METTL16 were revealed by CRISPR gene tiling scan, which will pave the way for the development of potential inhibitor(s).
CONCLUSIONS
Our findings highlight the crucial oncogenic role of METTL16 in promoting HCC pathogenesis and enhancing liver CSC self-renewal through augmenting mRNA translation efficiency.
Topics: Animals; Humans; Mice; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Self Renewal; Liver Neoplasms; Methyltransferases; Neoplastic Stem Cells; Protein Biosynthesis; Ribosomes; RNA
PubMed: 38302992
DOI: 10.1186/s13045-024-01526-9 -
BioRxiv : the Preprint Server For... Nov 2023During translation initiation, messenger RNA molecules must be identified and activated for loading into a ribosome. In this rate-limiting step, the heterotrimeric...
During translation initiation, messenger RNA molecules must be identified and activated for loading into a ribosome. In this rate-limiting step, the heterotrimeric protein eukaryotic initiation factor eIF4F must recognize and productively interact with the 7-methylguanosine cap at the 5' end of the messenger RNA and subsequently activate the message. Despite its fundamental, regulatory role in gene expression, the molecular events underlying cap recognition and messenger RNA activation remain mysterious. Here, we generate a unique, single-molecule fluorescence imaging system to interrogate the dynamics with which eIF4F discriminates productive and non-productive locations on full-length, native messenger RNA molecules. At the single-molecule level, we observe stochastic sampling of eIF4F along the length of the messenger RNA and identify allosteric communication between the eIF4F subunits which ultimately drive cap-recognition and subsequent activation of the message. Our experiments uncover novel functions for each subunit of eIF4F and we conclude by presenting a model for messenger RNA activation which precisely defines the composition of the activated message. This model provides a general framework for understanding how messenger RNA molecules may be discriminated from one another, and how other RNA-binding proteins may control the efficiency of translation initiation.
PubMed: 38014128
DOI: 10.1101/2023.11.15.567265 -
Seminars in Cell & Developmental Biology Feb 2024Cells entrust ribosomes with the critical task of identifying problematic mRNAs and facilitating their degradation. Ribosomes must communicate when they encounter and... (Review)
Review
Cells entrust ribosomes with the critical task of identifying problematic mRNAs and facilitating their degradation. Ribosomes must communicate when they encounter and stall on an aberrant mRNA, lest they expose the cell to toxic and disease-causing proteins, or they jeopardize ribosome homeostasis and cellular translation. In recent years, ribosomal ubiquitination has emerged as a central signaling step in this process, and proteomic studies across labs and experimental systems show a myriad of ubiquitination sites throughout the ribosome. Work from many labs zeroed in on ubiquitination in one region of the small ribosomal subunit as being functionally significant, with the balance and exact ubiquitination sites determined by stall type, E3 ubiquitin ligases, and deubiquitinases. This review discusses the current literature surrounding ribosomal ubiquitination during translational stress and considers its role in committing translational complexes to decay.
Topics: Ubiquitin; Proteomics; Saccharomyces cerevisiae; Ribosomes; Ubiquitination; RNA, Messenger; Protein Biosynthesis
PubMed: 36963992
DOI: 10.1016/j.semcdb.2023.03.009 -
Trends in Biochemical Sciences Jul 2023The mitochondrial ribosome (mitoribosome) is a multicomponent machine that has unique structural features. Biogenesis of the human mitoribosome includes correct... (Review)
Review
The mitochondrial ribosome (mitoribosome) is a multicomponent machine that has unique structural features. Biogenesis of the human mitoribosome includes correct maturation and folding of the mitochondria-encoded RNA components (12S and 16S mt-rRNAs, and mt-tRNAVal) and their assembly together with 82 nucleus-encoded mitoribosomal proteins. This complex process requires the coordinated action of multiple assembly factors. Recent advances in single-particle cryo-electron microscopy (cryo-EM) have provided detailed insights into the specific functions of several mitoribosome assembly factors and have defined their timing. In this review we summarize mitoribosomal small (mtSSU) and large subunit (mtLSU) biogenesis based on structural findings, and we discuss potential crosstalk between mtSSU and mtLSU assembly pathways as well as coordination between mitoribosome biogenesis and other processes involved in mitochondrial gene expression.
Topics: Humans; Cryoelectron Microscopy; Mitochondrial Ribosomes; RNA, Ribosomal, 16S; Mitochondrial Proteins; Ribosomal Proteins
PubMed: 37169615
DOI: 10.1016/j.tibs.2023.04.002 -
Cell Reports Nov 2023Oncogene-induced senescence (OIS) is a persistent anti-proliferative response that acts as a barrier against malignant transformation. During OIS, cells undergo dynamic...
Oncogene-induced senescence (OIS) is a persistent anti-proliferative response that acts as a barrier against malignant transformation. During OIS, cells undergo dynamic remodeling, which involves alterations in protein and organelle homeostasis through autophagy. Here, we show that ribosomes are selectively targeted for degradation by autophagy during OIS. By characterizing senescence-dependent alterations in the ribosomal interactome, we find that the deubiquitinase USP10 dissociates from the ribosome during the transition to OIS. This release of USP10 leads to an enhanced ribosome ubiquitination, particularly of small subunit proteins, including lysine 275 on RPS2. Both reinforcement of the USP10-ribosome interaction and mutation of RPS2 K275 abrogate ribosomal delivery to lysosomes without affecting bulk autophagy. We show that the selective recruitment of ubiquitinated ribosomes to autophagosomes is mediated by the p62 receptor. While ribophagy is not required for the establishment of senescence per se, it contributes to senescence-related metabolome alterations and facilitates the senescence-associated secretory phenotype.
Topics: Ribosomes; Ubiquitination; Ubiquitin; Autophagy; Oncogenes; Cellular Senescence
PubMed: 37930887
DOI: 10.1016/j.celrep.2023.113381 -
Trends in Cell Biology Sep 2023Most mitochondrial proteins are synthesized in the cytosol and transported into mitochondria by protein translocases. Yet, mitochondria contain their own genome and gene... (Review)
Review
Most mitochondrial proteins are synthesized in the cytosol and transported into mitochondria by protein translocases. Yet, mitochondria contain their own genome and gene expression system, which generates proteins that are inserted in the inner membrane by the oxidase assembly (OXA) insertase. OXA contributes to targeting proteins from both genetic origins. Recent data provides insights into how OXA cooperates with the mitochondrial ribosome during synthesis of mitochondrial-encoded proteins. A picture of OXA emerges in which it coordinates insertion of OXPHOS core subunits and their assembly into protein complexes but also participates in the biogenesis of select imported proteins. These functions position the OXA as a multifunctional protein insertase that facilitates protein transport, assembly, and stability at the inner membrane.
Topics: Humans; Oxidoreductases; Electron Transport Complex IV; Mitochondria; Mitochondrial Proteins; Carrier Proteins
PubMed: 36863885
DOI: 10.1016/j.tcb.2023.02.001 -
Nature Communications Sep 2023Neovascular age-related macular degeneration (nAMD), along with its clinical subtype known as polypoidal choroidal vasculopathy (PCV), are among the leading causes of...
Neovascular age-related macular degeneration (nAMD), along with its clinical subtype known as polypoidal choroidal vasculopathy (PCV), are among the leading causes of vision loss in elderly Asians. In a genome-wide association study (GWAS) comprising 3,128 nAMD (1,555 PCV and 1,573 typical nAMD), and 5,493 controls of East Asian ancestry, we identify twelve loci, of which four are novel ([Formula: see text]). Substantial genetic sharing between PCV and typical nAMD is noted (r = 0.666), whereas collagen extracellular matrix and fibrosis-related pathways are more pronounced for PCV. Whole-exome sequencing in 259 PCV patients revealed functional rare variants burden in collagen type I alpha 1 chain gene (COL1A1; [Formula: see text]) and potential enrichment of functional rare mutations at AMD-associated loci. At the GATA binding protein 5 (GATA5) locus, the most significant GWAS novel loci, the expressions of genes including laminin subunit alpha 5 (Lama5), mitochondrial ribosome associated GTPase 2 (Mtg2), and collagen type IX alpha 3 chain (Col9A3), are significantly induced during retinal angiogenesis and subretinal fibrosis in murine models. Furthermore, retinoic acid increased the expression of LAMA5 and MTG2 in vitro. Taken together, our data provide insights into the genetic basis of AMD pathogenesis in the Asian population.
Topics: Aged; Animals; Humans; Mice; Asian; East Asian People; Extracellular Matrix; Genome-Wide Association Study; Macular Degeneration; Polypoidal Choroidal Vasculopathy; Disease Models, Animal
PubMed: 37696869
DOI: 10.1038/s41467-023-41256-z -
Nucleic Acids Research Sep 2023Archaeal ribosomes have many domain-specific features; however, our understanding of these structures is limited. We present 10 cryo-electron microscopy (cryo-EM)...
Archaeal ribosomes have many domain-specific features; however, our understanding of these structures is limited. We present 10 cryo-electron microscopy (cryo-EM) structures of the archaeal ribosome from crenarchaeota Sulfolobus acidocaldarius (Sac) at 2.7-5.7 Å resolution. We observed unstable conformations of H68 and h44 of ribosomal RNA (rRNA) in the subunit structures, which may interfere with subunit association. These subunit structures provided models for 12 rRNA expansion segments and 3 novel r-proteins. Furthermore, the 50S-aRF1 complex structure showed the unique domain orientation of aRF1, possibly explaining P-site transfer RNA (tRNA) release after translation termination. Sac 70S complexes were captured in seven distinct steps of the tRNA translocation reaction, confirming conserved structural features during archaeal ribosome translocation. In aEF2-engaged 70S ribosome complexes, 3D classification of cryo-EM data based on 30S head domain identified two new translocation intermediates with 30S head domain tilted 5-6° enabling its disengagement from the translocated tRNA and its release post-translocation. Additionally, we observed conformational changes to aEF2 during ribosome binding and switching from three different states. Our structural and biochemical data provide new insights into archaeal translation and ribosome translocation.
Topics: Cryoelectron Microscopy; Ribosomal Proteins; Ribosomes; RNA, Ribosomal; RNA, Transfer; Sulfolobus acidocaldarius
PubMed: 37604686
DOI: 10.1093/nar/gkad661