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Molecular Cell Oct 2021The human genome encodes tens of thousands circular RNAs (circRNAs) with mostly unknown functions. Circular RNAs require internal ribosome entry sites (IRES) if they are...
The human genome encodes tens of thousands circular RNAs (circRNAs) with mostly unknown functions. Circular RNAs require internal ribosome entry sites (IRES) if they are to undergo translation without a 5' cap. Here, we develop a high-throughput screen to systematically discover RNA sequences that can direct circRNA translation in human cells. We identify more than 17,000 endogenous and synthetic sequences as candidate circRNA IRES. 18S rRNA complementarity and a structured RNA element positioned on the IRES are important for driving circRNA translation. Ribosome profiling and peptidomic analyses show extensive IRES-ribosome association, hundreds of circRNA-encoded proteins with tissue-specific distribution, and antigen presentation. We find that circFGFR1p, a protein encoded by circFGFR1 that is downregulated in cancer, functions as a negative regulator of FGFR1 oncoprotein to suppress cell growth during stress. Systematic identification of circRNA IRES elements may provide important links among circRNA regulation, biological function, and disease.
Topics: Cell Proliferation; Gene Expression Regulation, Neoplastic; HEK293 Cells; HeLa Cells; Humans; Internal Ribosome Entry Sites; Mutation; Neoplasms; Nucleic Acid Conformation; Protein Biosynthesis; RNA, Circular; Receptor, Fibroblast Growth Factor, Type 1; Ribosome Subunits; Structure-Activity Relationship
PubMed: 34437836
DOI: 10.1016/j.molcel.2021.07.042 -
Cell May 2023Ribosome production is vital for every cell, and failure causes human diseases. It is driven by ∼200 assembly factors functioning along an ordered pathway from the...
Ribosome production is vital for every cell, and failure causes human diseases. It is driven by ∼200 assembly factors functioning along an ordered pathway from the nucleolus to the cytoplasm. Structural snapshots of biogenesis intermediates from the earliest 90S pre-ribosomes to mature 40S subunits unravel the mechanisms of small ribosome synthesis. To view this SnapShot, open or download the PDF.
Topics: Humans; Cell Nucleolus; Eukaryotic Cells; Ribosomal Proteins; Ribosome Subunits, Small, Eukaryotic; Ribosomes
PubMed: 37172570
DOI: 10.1016/j.cell.2023.04.030 -
Nucleic Acids Research Jul 2022Human ribosomes have long been thought to be uniform factories with little regulatory function. Accumulating evidence emphasizes the heterogeneity of ribosomal protein...
Human ribosomes have long been thought to be uniform factories with little regulatory function. Accumulating evidence emphasizes the heterogeneity of ribosomal protein (RP) expression in specific cellular functions and development. However, a systematic understanding of functional relevance of RPs is lacking. Here, we surveyed translational and transcriptional changes after individual knockdown of 75 RPs, 44 from the large subunit (60S) and 31 from the small subunit (40S), by Ribo-seq and RNA-seq analyses. Deficiency of individual RPs altered specific subsets of genes transcriptionally and translationally. RP genes were under cotranslational regulation upon ribosomal stress, and deficiency of the 60S RPs and the 40S RPs had opposite effects. RP deficiency altered the expression of genes related to eight major functional classes, including the cell cycle, cellular metabolism, signal transduction and development. 60S RP deficiency led to greater inhibitory effects on cell growth than did 40S RP deficiency, through P53 signaling. Particularly, we showed that eS8/RPS8 deficiency stimulated apoptosis while eL13/RPL13 or eL18/RPL18 deficiency promoted senescence. We also validated the phenotypic impacts of uL5/RPL11 and eL15/RPL15 deficiency on retina development and angiogenesis, respectively. Overall, our study provides a valuable resource for and novel insights into ribosome regulation in cellular activities, development and diseases.
Topics: Gene Knockdown Techniques; Humans; Protein Biosynthesis; Ribosomal Proteins; Ribosome Subunits, Large, Eukaryotic; Ribosome Subunits, Small, Eukaryotic; Transcription, Genetic
PubMed: 35137207
DOI: 10.1093/nar/gkac053 -
Science (New York, N.Y.) Sep 2020Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1), which suppresses host gene expression by ribosome association. Here, we show that Nsp1 from SARS-CoV-2 binds to the 40 ribosomal subunit, resulting in shutdown of messenger RNA (mRNA) translation both in vitro and in cells. Structural analysis by cryo-electron microscopy of in vitro-reconstituted Nsp1-40 and various native Nsp1-40 and -80 complexes revealed that the Nsp1 C terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks retinoic acid-inducible gene I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.
Topics: Betacoronavirus; Binding Sites; COVID-19; Coronavirus Infections; Cryoelectron Microscopy; DEAD Box Protein 58; Humans; Immune Evasion; Immunity, Innate; Interferon-beta; Models, Molecular; Pandemics; Pneumonia, Viral; Protein Binding; Protein Biosynthesis; Protein Domains; Protein Interaction Domains and Motifs; Protein Structure, Secondary; RNA, Messenger; Receptors, Immunologic; Ribosome Subunits, Small, Eukaryotic; SARS-CoV-2; Viral Nonstructural Proteins
PubMed: 32680882
DOI: 10.1126/science.abc8665 -
Nature Reviews. Molecular Cell Biology Feb 2019In the past 25 years, genetic and biochemical analyses of ribosome assembly in yeast have identified most of the factors that participate in this complex pathway and... (Review)
Review
In the past 25 years, genetic and biochemical analyses of ribosome assembly in yeast have identified most of the factors that participate in this complex pathway and have generated models for the mechanisms driving the assembly. More recently, the publication of numerous cryo-electron microscopy structures of yeast ribosome assembly intermediates has provided near-atomic resolution snapshots of ribosome precursor particles. Satisfyingly, these structural data support the genetic and biochemical models and provide additional mechanistic insight into ribosome assembly. In this Review, we discuss the mechanisms of assembly of the yeast small ribosomal subunit and large ribosomal subunit in the nucleolus, nucleus and cytoplasm. Particular emphasis is placed on concepts such as the mechanisms of RNA compaction, the functions of molecular switches and molecular mimicry, the irreversibility of assembly checkpoints and the roles of structural and functional proofreading of pre-ribosomal particles.
Topics: Animals; Cell Nucleus; Cryoelectron Microscopy; Cytoplasm; Humans; RNA; Ribosome Subunits
PubMed: 30467428
DOI: 10.1038/s41580-018-0078-y -
The Journal of Antimicrobial... Apr 2020This article describes 20 years of research that investigated a second novel target for ribosomal antibiotics, the biogenesis of the two subunits. Over that period, we... (Review)
Review
This article describes 20 years of research that investigated a second novel target for ribosomal antibiotics, the biogenesis of the two subunits. Over that period, we have examined the effect of 52 different antibiotics on ribosomal subunit formation in six different microorganisms. Most of the antimicrobials we have studied are specific, preventing the formation of only the subunit to which they bind. A few interesting exceptions have also been observed. Forty-one research publications and a book chapter have resulted from this investigation. This review will describe the methodology we used and the fit of our results to a hypothetical model. The model predicts that inhibition of subunit assembly and translation are equivalent targets for most of the antibiotics we have investigated.
Topics: Anti-Bacterial Agents; Bacteria; Protein Biosynthesis; Ribosomal Proteins; Ribosome Subunits; Ribosomes
PubMed: 31942624
DOI: 10.1093/jac/dkz544 -
Nature Jun 2022Mitoribosomes are essential for the synthesis and maintenance of bioenergetic proteins. Here we use cryo-electron microscopy to determine a series of the small...
Mitoribosomes are essential for the synthesis and maintenance of bioenergetic proteins. Here we use cryo-electron microscopy to determine a series of the small mitoribosomal subunit (SSU) intermediates in complex with auxiliary factors, revealing a sequential assembly mechanism. The methyltransferase TFB1M binds to partially unfolded rRNA h45 that is promoted by RBFA, while the mRNA channel is blocked. This enables binding of METTL15 that promotes further rRNA maturation and a large conformational change of RBFA. The new conformation allows initiation factor mtIF3 to already occupy the subunit interface during the assembly. Finally, the mitochondria-specific ribosomal protein mS37 (ref. ) outcompetes RBFA to complete the assembly with the SSU-mS37-mtIF3 complex that proceeds towards mtIF2 binding and translation initiation. Our results explain how the action of step-specific factors modulate the dynamic assembly of the SSU, and adaptation of a unique protein, mS37, links the assembly to initiation to establish the catalytic human mitoribosome.
Topics: Humans; Cryoelectron Microscopy; DNA-Binding Proteins; Eukaryotic Initiation Factors; Mitochondria; Mitochondrial Proteins; Mitochondrial Ribosomes; Ribosomal Proteins; Ribosome Subunits, Small; RNA, Ribosomal; RNA-Binding Proteins; Transcription Factors
PubMed: 35676484
DOI: 10.1038/s41586-022-04795-x -
Molecules (Basel, Switzerland) Nov 2016Ribosome-inactivating proteins (RIPs) including ricin, Shiga toxin, and trichosanthin, are RNA -glycosidases that depurinate a specific adenine residue (A-4324 in rat... (Review)
Review
Ribosome-inactivating proteins (RIPs) including ricin, Shiga toxin, and trichosanthin, are RNA -glycosidases that depurinate a specific adenine residue (A-4324 in rat 28S ribosomal RNA, rRNA) in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. RIPs are grouped into three types according to the number of subunits and the organization of the precursor sequences. RIPs are two-domain proteins, with the active site located in the cleft between the N- and C-terminal domains. It has been found that the basic surface residues of the RIPs promote rapid and specific targeting to the ribosome and a number of RIPs have been shown to interact with the C-terminal regions of the P proteins of the ribosome. At present, the structural basis for the interaction of trichosanthin and ricin-A chain toward P2 peptide is known. This review surveys the structural features of the representative RIPs and discusses how they approach and interact with the ribosome.
Topics: Catalytic Domain; Models, Molecular; Molecular Structure; Protein Binding; Protein Interaction Domains and Motifs; Ribosome Inactivating Proteins; Ribosome Subunits; Ribosomes; Structure-Activity Relationship; Substrate Specificity
PubMed: 27879643
DOI: 10.3390/molecules21111588 -
Nature Communications May 2023In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal...
In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal subunit recycling. Despite its importance for translation efficiency the exact role and structural basis for this translational reset is poorly understood. Here, structural analysis by cryo-electron microscopy of native and reconstituted Otu2-bound ribosomal complexes reveals that Otu2 engages 40S subunits mainly between ribosome recycling and initiation stages. Otu2 binds to several sites on the intersubunit surface of the 40S that are not occupied by any other 40S-binding factors. This binding mode explains the discrimination against 80S ribosomes via the largely helical N-terminal domain of Otu2 as well as the specificity for mono-ubiquitinated eS7 on 40S. Collectively, this study reveals mechanistic insights into the Otu2-driven deubiquitination steps for translational reset during ribosome recycling/(re)initiation.
Topics: Cryoelectron Microscopy; Protein Biosynthesis; Ribosomal Proteins; Ribosome Subunits, Small, Eukaryotic; Ribosomes
PubMed: 37169754
DOI: 10.1038/s41467-023-38161-w -
International Journal of Molecular... Mar 2020Translation of the hepatitis C virus (HCV) RNA genome is regulated by the internal ribosome entry site (IRES), located in the 5'-untranslated region (5'UTR) and part of... (Review)
Review
Translation of the hepatitis C virus (HCV) RNA genome is regulated by the internal ribosome entry site (IRES), located in the 5'-untranslated region (5'UTR) and part of the core protein coding sequence, and by the 3'UTR. The 5'UTR has some highly conserved structural regions, while others can assume different conformations. The IRES can bind to the ribosomal 40S subunit with high affinity without any other factors. Nevertheless, IRES activity is modulated by additional sequences in the viral genome, including the 3'UTR and the -acting replication element (CRE). Canonical translation initiation factors (eIFs) are involved in HCV translation initiation, including eIF3, eIF2, eIF1A, eIF5, and eIF5B. Alternatively, under stress conditions and limited eIF2-Met-tRNA availability, alternative initiation factors such as eIF2D, eIF2A, and eIF5B can substitute for eIF2 to allow HCV translation even when cellular mRNA translation is downregulated. In addition, several IRES trans-acting factors (ITAFs) modulate IRES activity by building large networks of RNA-protein and protein-protein interactions, also connecting 5'- and 3'-ends of the viral RNA. Moreover, some ITAFs can act as RNA chaperones that help to position the viral AUG start codon in the ribosomal 40S subunit entry channel. Finally, the liver-specific microRNA-122 (miR-122) stimulates HCV IRES-dependent translation, most likely by stabilizing a certain structure of the IRES that is required for initiation.
Topics: 3' Untranslated Regions; 5' Untranslated Regions; Codon, Initiator; Eukaryotic Initiation Factor-2; Eukaryotic Initiation Factor-3; Eukaryotic Initiation Factors; Genome, Viral; Hepacivirus; Hepatitis C; Humans; Internal Ribosome Entry Sites; MicroRNAs; Models, Molecular; Peptide Chain Initiation, Translational; Protein Biosynthesis; RNA, Viral; Ribosome Subunits, Small, Eukaryotic; Ribosomes
PubMed: 32230899
DOI: 10.3390/ijms21072328