RESUMO
How eukaryotic ribosomes traverse messenger RNA (mRNA) leader sequences to search for protein-synthesis start sites remains one of the most mysterious aspects of translation and its regulation. While the search process is conventionally described by a linear "scanning" model, its exquisitely dynamic nature has restricted detailed mechanistic study. Here, we observed single Saccharomyces cerevisiae ribosomal scanning complexes in real time, finding that they scan diverse mRNA leaders at a rate of 10 to 20 nt s-1. We show that specific binding of a protein to its mRNA leader sequence substantially arrests scanning. Conversely, impairing scanning-complex guanosine 5'-triphosphate hydrolysis results in native start-site bypass. Our results illustrate an mRNA-centric, kinetically controlled regulatory model where the ribosomal pre-initiation complex amplifies a nuanced energetic landscape to regulate scanning and start-site selection fidelity.
Assuntos
RNA Mensageiro , Ribossomos , Saccharomyces cerevisiae , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Imagem Individual de Molécula/métodos , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Guanosina Trifosfato/metabolismoRESUMO
BACKGROUND: Several computational and mathematical models of protein synthesis have been explored to accomplish the quantitative analysis of protein synthesis components and polysome structure. The effect of gene sequence (coding and non-coding region) in protein synthesis, mutation in gene sequence, and functional model of ribosome needs to be explored to investigate the relationship among protein synthesis components further. Ribosomal computing is implemented by imitating the functional property of protein synthesis. RESULT: In the proposed work, a general framework of ribosomal computing is demonstrated by developing a computational model to present the relationship between biological details of protein synthesis and computing principles. Here, mathematical abstractions are chosen carefully without probing into intricate chemical details of the micro-operations of protein synthesis for ease of understanding. This model demonstrates the cause and effect of ribosome stalling during protein synthesis and the relationship between functional protein and gene sequence. Moreover, it also reveals the computing nature of ribosome molecules and other protein synthesis components. The effect of gene mutation on protein synthesis is also explored in this model. CONCLUSION: The computational model for ribosomal computing is implemented in this work. The proposed model demonstrates the relationship among gene sequences and protein synthesis components. This model also helps to implement a simulation environment (a simulator) for generating protein chains from gene sequences and can spot the problem during protein synthesis. Thus, this simulator can identify a disease that can happen due to a protein synthesis problem and suggest precautions for it.
Assuntos
Biologia Computacional , Biossíntese de Proteínas , Ribossomos , Ribossomos/metabolismo , Biologia Computacional/métodos , Simulação por Computador , MutaçãoRESUMO
Epithelial-to-mesenchymal transition (EMT) contributes significantly to chemotherapy resistance and remains a critical challenge in treating advanced breast cancer. The complexity of EMT, involving redundant pro-EMT signaling pathways and its paradox reversal process, mesenchymal-to-epithelial transition (MET), has hindered the development of effective treatments. In this study, we utilized a Tri-PyMT EMT lineage-tracing model in mice and single-cell RNA sequencing (scRNA-seq) to comprehensively analyze the EMT status of tumor cells. Our findings revealed elevated ribosome biogenesis (RiBi) during the transitioning phases of both EMT and MET processes. RiBi and its subsequent nascent protein synthesis mediated by ERK and mTOR signalings are essential for EMT/MET completion. Importantly, inhibiting excessive RiBi genetically or pharmacologically impaired the EMT/MET capability of tumor cells. Combining RiBi inhibition with chemotherapy drugs synergistically reduced metastatic outgrowth of epithelial and mesenchymal tumor cells under chemotherapies. Our study suggests that targeting the RiBi pathway presents a promising strategy for treating patients with advanced breast cancer.
Although there have been considerable improvements in breast cancer treatments over the years, there are still many patients whose cancerous cells become resistant to treatments, including chemotherapy. Several different factors can contribute to resistance to chemotherapy, but one important change is the epithelial-to-mesenchymal transition (or EMT for short). During this transition, breast cancer cells become more aggressive, and more able to metastasize and spread to other parts of the body. Cells can also go through the reverse process called the mesenchymal-to-epithelial transition (or MET for short). Together, EMT and MET help breast cancer cells become resilient to treatment. However, it was not clear if these transitions shared a mechanism or pathway that could be targeted as a way to make cancer treatments more effective. To investigate, Ban, Zou et al. studied breast cancer cells from mice which had been labelled with fluorescent proteins that indicated whether a cell had ever transitioned between an epithelial and mesenchymal state. Various genetic experiments revealed that breast cancer cells in the EMT or MET phase made a lot more ribosomes, molecules that are vital for producing new proteins. Ban, Zhou et al. found that blocking the production of ribosomes (using drugs or genetic tools) prevented the cells from undergoing both EMT and MET. Further experiments showed that when mice with breast cancer were treated with a standard chemotherapy treatment plus an anti-ribosome drug, this reduced the number and size of tumors that had metastasized to the lung. This suggests that blocking ribosome production makes breast cancer cells undergoing EMT and/or MET less resistant to chemotherapy. Future studies will have to ascertain whether these findings also apply to patients with breast cancer. In particular, one of the drugs used to block ribosome production in this study is in early-phase clinical trials, so future trials may be able to assess the drug's effect in combination with chemotherapies.
Assuntos
Neoplasias da Mama , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Ribossomos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Camundongos , Feminino , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Humanos , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Biogênese de Organelas , Transdução de Sinais/efeitos dos fármacosRESUMO
In this issue of Cell Genomics, Rothschild et al.1 reveal how ribosomal RNA diversity impacts ribosome structure and its implications for health and disease. Their innovative methodologies uncover distinct ribosome subtypes with significant structural variations and expression patterns. This work reveals connections to tissue-specific biology and cancer, positing new research avenues.
Assuntos
Ribossomos , Ribossomos/metabolismo , Ribossomos/genética , Humanos , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Neoplasias/genética , Neoplasias/patologiaRESUMO
Ribosomes are emerging as direct regulators of gene expression, with ribosome-associated proteins (RAPs) allowing ribosomes to modulate translation. Nevertheless, a lack of technologies to enrich RAPs across sample types has prevented systematic analysis of RAP identities, dynamics, and functions. We have developed a label-free methodology called RAPIDASH to enrich ribosomes and RAPs from any sample. We applied RAPIDASH to mouse embryonic tissues and identified hundreds of potential RAPs, including Dhx30 and Llph, two forebrain RAPs important for neurodevelopment. We identified a critical role of LLPH in neural development linked to the translation of genes with long coding sequences. In addition, we showed that RAPIDASH can identify ribosome changes in cancer cells. Finally, we characterized ribosome composition remodeling during immune cell activation and observed extensive changes post-stimulation. RAPIDASH has therefore enabled the discovery of RAPs in multiple cell types, tissues, and stimuli and is adaptable to characterize ribosome remodeling in several contexts.
Assuntos
Macrófagos , Proteínas Ribossômicas , Ribossomos , Animais , Ribossomos/metabolismo , Ribossomos/genética , Camundongos , Humanos , Macrófagos/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Biossíntese de Proteínas , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Regulação da Expressão Gênica no Desenvolvimento , Linhagem Celular Tumoral , Camundongos Endogâmicos C57BLRESUMO
Caliciviruses have positive-sense RNA genomes, typically with short 5'-untranslated regions (5'UTRs) that precede the long open reading frame 1 (ORF1). Exceptionally, some avian caliciviruses have long 5'UTRs containing a picornavirus-like internal ribosomal entry site (IRES), which was likely acquired by horizontal gene transfer. Here, we identified numerous additional avian calicivirus genomes with IRESs, predominantly type 2, and determined that many of these genomes contain a ~200-300 codon-long ORF (designated ORF1*) that overlaps the 5'-terminal region of ORF1. The activity of representative type 2 IRESs from grey teal calicivirus (GTCV) and Caliciviridae sp. isolate yc-13 (RaCV1) was confirmed by in vitro translation. Toeprinting showed that in cell-free extracts and in vitro reconstituted reactions, ribosomal initiation complexes assembled on the ORF1* initiation codon and at one or two AUG codons in ORF1 at the 3'-border and/or downstream of the IRES. Initiation at all three sites required eIF4A and eIF4G, which bound to a conserved region of the IRES; initiation on the ORF1* and principal ORF1 initiation codons involved eIF1/eIF1A-dependent scanning from the IRES's 3'-border. Initiation on these IRESs was enhanced by the IRES trans-acting factors (ITAFs) Ebp1/ITAF45, which bound to the apical subdomain Id of the IRES, and PTB (GTCV) or PCBP2 (RaCV1).
Assuntos
Caliciviridae , Genoma Viral , Sítios Internos de Entrada Ribossomal , Fases de Leitura Aberta , Biossíntese de Proteínas , Animais , Sítios Internos de Entrada Ribossomal/genética , Caliciviridae/genética , RNA Viral/genética , Regiões 5' não Traduzidas/genética , Ribossomos/metabolismo , Ribossomos/genéticaRESUMO
Infection with Campylobacter jejuni is the major cause of human gastroenteritis in the United States and Europe, leading to debilitating autoimmune sequelae in many cases. While considerable progress has been made in detailing the infectious cycle of C. jejuni, a full understanding of the molecular mechanisms responsible for virulence remains to be elucidated. Here, we apply a novel approach by modulating protein expression on the pathogen's ribosomes by inactivating a highly conserved rRNA methyltransferase. Loss of the RsmA methyltransferase results in a more motile strain with greater adhesive and cell-invasive properties. These phenotypical effects correlate with enhanced expression of specific proteins related to flagellar formation and function, together with enzymes involved in cell wall/membrane and amino acid synthesis. Despite the enhancement of certain virulent traits, the null strain grows poorly on minimal media and is rapidly out-competed by the wild-type strain. Complementation with an active copy of the rsmA gene rescues most of the traits changed in the mutant. However, the complemented strain overexpresses rsmA and displays new flaws, including loss of the spiral cell shape, which is distinctive for C. jejuni. Proteins linked with altered virulence and morphology are identified here by mass spectrometry proteomic analyses of the strains.
Assuntos
Proteínas de Bactérias , Campylobacter jejuni , Metiltransferases , Ribossomos , Campylobacter jejuni/patogenicidade , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Ribossomos/metabolismo , Ribossomos/genética , Virulência/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Metiltransferases/metabolismo , Metiltransferases/genética , Metilação , Regulação Bacteriana da Expressão Gênica , Humanos , Infecções por Campylobacter/microbiologia , Proteômica/métodosRESUMO
SETD8 is a methyltransferase that is overexpressed in several cancers, which monomethylates H4K20 as well as other non-histone targets such as PCNA or p53. We here report novel SETD8 inhibitors, which were discovered while trying to identify chemicals that prevent 53BP1 foci formation, an event mediated by H4K20 methylation. Consistent with previous reports, SETD8 inhibitors induce p53 expression, although they are equally toxic for p53 proficient or deficient cells. Thermal stability proteomics revealed that the compounds had a particular impact on nucleoli, which was confirmed by fluorescent and electron microscopy. Similarly, Setd8 deletion generated nucleolar stress and impaired ribosome biogenesis, supporting that this was an on-target effect of SETD8 inhibitors. Furthermore, a genome-wide CRISPR screen identified an enrichment of nucleolar factors among those modulating the toxicity of SETD8 inhibitors. Accordingly, the toxicity of SETD8 inhibition correlated with MYC or mTOR activity, key regulators of ribosome biogenesis. Together, our study provides a new class of SETD8 inhibitors and a novel biomarker to identify tumors most likely to respond to this therapy.
Assuntos
Histona-Lisina N-Metiltransferase , Ribossomos , Humanos , Ribossomos/metabolismo , Ribossomos/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Linhagem Celular Tumoral , Nucléolo Celular/metabolismo , Nucléolo Celular/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/metabolismo , Neoplasias/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
The emergence of the "super fungus" Candida auris poses a significant threat to human health, given its multidrug resistance and high mortality rates. Therefore, developing a new antifungal strategy is necessary. Our previous research showed that Baicalein (BE), a key bioactive compound from the dried root of the perennial herb Scutellaria baicalensis Georgi, has strong fungistatic properties against C. auris. Nevertheless, the antifungal activity of BE against C. auris and its mechanism of action requires further investigation. In this study, we explored how BE affects this fungus using various techniques, including scanning electron microscopy (SEM), Annexin V-FITC apoptosis detection, CaspACE FITC-VAD-FMK In Situ Marker, reactive oxygen species (ROS) assay, singlet oxygen sensor green (SOSG) fluorescent probe, enhanced mitochondrial membrane potential (MMP) assay with JC-1, DAPI staining, TUNEL assay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Our findings revealed that BE induced several apoptotic features, including phosphatidylserine (PS) externalization, metacaspase activation, nuclear condensation and DNA fragmentation. BE also increased intracellular ROS levels and altered mitochondrial functions. Additionally, transcriptomic analysis and RT-qPCR validation indicated that BE may induce apoptosis in C. auris by affecting ribosome-related pathways, suggesting that ribosomes could be new targets for antifungal agents, in addition to cell walls, membranes, and DNA. This study emphasizes the antifungal activity and mechanism of BE against C. auris, offering a promising treatment strategy for C. auris infection.
Assuntos
Antifúngicos , Apoptose , Candida , Flavanonas , Potencial da Membrana Mitocondrial , Espécies Reativas de Oxigênio , Ribossomos , Flavanonas/farmacologia , Apoptose/efeitos dos fármacos , Candida/efeitos dos fármacos , Antifúngicos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Testes de Sensibilidade Microbiana , HumanosRESUMO
Abnormal biosynthesis of spliceosomes and ribosomes can lead to their dysfunction, which in turn disrupts protein synthesis and results in various diseases. While genetic factors have been extensively studied, our understanding of how environmental compounds interfere with spliceosome and ribosome biosynthesis remains limited. In the present study, we employed a Reduced Transcriptome Analysis (RTA) approach, integrating large-scale transcriptome data sets of zebrafish and compiling a specific zebrafish gene panel focusing on the spliceosome and ribosome, to elucidate the potential disruptors targeting their biosynthesis. Transcriptomic data sets for 118 environmental substances and 1400 related gene expression profiles were integrated resulting in 513 exposure signatures. Among these substances, several categories including PCB126, transition metals Lanthanum (La) and praseodymium (Pr), heavy metals Cd2+ and AgNO3 and atrazine were highlighted for inducing the significant transcriptional alterations. Furthermore, we found that the transcriptional patterns were distinct between categories, yet overlapping patterns were generally observed within each group. For instance, over 82 % differentially expressed ribosomal genes were shared between La and Pr within the equivalent concentration range. Additionally, transcriptional complexities were also evident across various organs and developmental stages of zebrafish, with notable differences in the inhibition of the transcription of various spliceosome subunits. Overall, our results provide novel insights into the understanding of the adverse effects of environmental compounds, thereby contributing to their environmental risk assessments.
Assuntos
Ribossomos , Spliceossomos , Transcriptoma , Peixe-Zebra , Peixe-Zebra/genética , Animais , Spliceossomos/metabolismo , Spliceossomos/efeitos dos fármacos , Ribossomos/metabolismo , Perfilação da Expressão Gênica , Poluentes Químicos da Água/toxicidadeRESUMO
Modification of guanosine to N7-methylguanosine (m7G) in the variable loop region of tRNA is catalyzed by the METTL1/WDR4 heterodimer and stabilizes target tRNA. Here, we reveal essential functions of Mettl1 in Drosophila fertility. Knockout of Mettl1 (Mettl1-KO) causes no major effect on the development of non-gonadal tissues, but abolishes the production of elongated spermatids and mature sperm, which is fully rescued by expression of a Mettl1-transgene, but not a catalytic-dead Mettl1 transgene. This demonstrates that Mettl1-dependent m7G is required for spermatogenesis. Mettl1-KO results in a loss of m7G modification on a subset of tRNAs and decreased tRNA abundance. Ribosome profiling shows that Mettl1-KO led to ribosomes stalling at codons decoded by tRNAs that were reduced in abundance. Mettl1-KO also significantly reduces the translation efficiency of genes involved in elongated spermatid formation and sperm stability. Germ cell-specific expression of Mettl1 rescues disrupted m7G tRNA modification and tRNA abundance in Mettl1-KO testes but not in non-gonadal tissues. Ribosome stalling is much less detectable in non-gonadal tissues than in Mettl1-KO testes. These findings reveal a developmental role for m7G tRNA modification and indicate that m7G modification-dependent tRNA abundance differs among tissues.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Fertilidade , RNA de Transferência , Espermatogênese , Animais , Espermatogênese/genética , Masculino , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência/genética , Fertilidade/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Guanosina/metabolismo , Guanosina/análogos & derivados , Metiltransferases/metabolismo , Metiltransferases/genética , Espermatozoides/metabolismo , Ribossomos/metabolismo , Espermátides/metabolismo , Testículo/metabolismo , Técnicas de Inativação de GenesRESUMO
Eukaryotic cells encode thousands of secretory and membrane proteins, many of which are cotranslationally translocated into the endoplasmic reticulum (ER). Nascent polypeptides entering the ER encounter a network of molecular chaperones and enzymes that facilitate their folding. A rate-limiting step for some proteins is the trans-to-cis isomerization of the peptide bond between proline and the residue preceding it. The human ER contains six prolyl isomerases, but the function, organization, and substrate range of these proteins is not clear. Here we show that the metazoan-specific, prolyl isomerase FKBP11 binds to ribosome-translocon complexes (RTCs) in the ER membrane, dependent on its single transmembrane domain and a conserved, positively charged region at its cytosolic C-terminus. High-throughput mRNA sequencing shows selective engagement with ribosomes synthesizing secretory and membrane proteins with long translocated segments, and functional analysis shows reduced stability of two such proteins, EpCAM and PTTG1IP, in cells depleted of FKBP11. We propose that FKBP11 is a translocon accessory factor that acts on a broad range of soluble secretory and transmembrane proteins during their synthesis at the ER.
Assuntos
Retículo Endoplasmático , Peptidilprolil Isomerase , Transporte Proteico , Ribossomos , Proteínas de Ligação a Tacrolimo , Humanos , Proteínas de Ligação a Tacrolimo/metabolismo , Retículo Endoplasmático/metabolismo , Ribossomos/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas de Membrana/metabolismo , Biossíntese de Proteínas , Células HEK293 , Células HeLa , Dobramento de Proteína , Ligação ProteicaRESUMO
Accuracy and efficiency are fundamental to mRNA translation. Codon usage bias is widespread across species. Despite the long-standing association between optimized codon usage and improved translation, our understanding of its evolutionary basis and functional effects remains limited. Drosophila is widely used to study codon usage bias, but genome-scale experimental data are scarce. Using high-resolution mass spectrometry data from Drosophila melanogaster, we show that optimal codons have lower translation errors than nonoptimal codons after accounting for these biases. Genomic-scale analysis of ribosome profiling data shows that optimal codons are translated more rapidly than nonoptimal codons. Although we find no long-term selection favoring synonymous mutations in D. melanogaster after diverging from D. simulans, we identify signatures of positive selection driving codon optimization in the D. melanogaster population. These findings expand our understanding of the functional consequences of codon optimization and serve as a foundation for future investigations.
Assuntos
Uso do Códon , Drosophila melanogaster , Biossíntese de Proteínas , Animais , Drosophila melanogaster/genética , Genoma de Inseto , Ribossomos/metabolismo , Ribossomos/genética , Códon/genética , Evolução Molecular , Seleção Genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Cancer cells rely on high ribosome production to sustain their proliferation rate. Many chemotherapies impede ribosome production which is perceived by cells as "nucleolar stress" (NS), triggering p53-dependent and independent pathways leading to cell cycle arrest and/or apoptosis. The 5S ribonucleoprotein (RNP) particle, a sub-ribosomal particle, is instrumental to NS response. Upon ribosome assembly defects, the 5S RNP accumulates as free form. This free form is able to sequester and inhibit MDM2, thus promoting p53 stabilization. To investigate how cancer cells can resist to NS, here we purify free 5S RNP and uncover an interaction partner, SURF2. Functional characterization of SURF2 shows that its depletion increases cellular sensitivity to NS, while its overexpression promotes their resistance to it. Consistently, SURF2 is overexpressed in many cancers and its expression level is an independent marker of prognosis for adrenocortical cancer. Our data demonstrate that SURF2 buffers free 5S RNP particles, and can modulate their activity, paving the way for the research of new molecules that can finely tune the response to nucleolar stress in the framework of cancer therapies.
Assuntos
Nucléolo Celular , Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p53 , Humanos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/genética , Nucléolo Celular/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Estresse Fisiológico/efeitos dos fármacos , Ribossomos/metabolismo , Apoptose/efeitos dos fármacosRESUMO
Small-molecule compounds that elicit mRNA-selective translation repression have attracted interest due to their potential for expansion of druggable space. However, only a limited number of examples have been reported to date. Here, we show that desmethyl desamino pateamine A (DMDA-PatA) represses translation in an mRNA-selective manner by clamping eIF4A, a DEAD-box RNA-binding protein, onto GNG motifs. By systematically comparing multiple eIF4A inhibitors by ribosome profiling, we found that DMDA-PatA has unique mRNA selectivity for translation repression. Unbiased Bind-n-Seq reveals that DMDA-PatA-targeted eIF4A exhibits a preference for GNG motifs in an ATP-independent manner. This unusual RNA binding sterically hinders scanning by 40S ribosomes. A combination of classical molecular dynamics simulations and quantum chemical calculations, and the subsequent development of an inactive DMDA-PatA derivative reveals that the positive charge of the tertiary amine on the trienyl arm induces G selectivity. Moreover, we identified that DDX3, another DEAD-box protein, is an alternative DMDA-PatA target with the same effects on eIF4A. Our results provide an example of the sequence-selective anchoring of RNA-binding proteins and the mRNA-selective inhibition of protein synthesis by small-molecule compounds.
Assuntos
RNA Helicases DEAD-box , Fator de Iniciação 4A em Eucariotos , Biossíntese de Proteínas , RNA Mensageiro , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Simulação de Dinâmica Molecular , Ribossomos/metabolismo , Motivos de Nucleotídeos , Ligação Proteica , Células HEK293 , Compostos de Epóxi , Tiazóis , MacrolídeosRESUMO
Ribosome has been considered as the fundamental macromolecular machine involved in protein synthesis in both prokaryotic and eukaryotic cells. This protein synthesis machinery consists of several rRNAs and numerous proteins. All of these factors are synthesized, translocated and assembled in a tightly regulated process known as ribosome biogenesis. Any impairment in this process causes development of several diseases like cancer. According to growing evidences, cancer cells display alteration of several ribosomal proteins. Besides, most of them are considered as key molecules involved in ribosome biogenesis, suggesting a correlation between those proteins and formation of ribosomes. Albeit, defective ribosome biogenesis in several cancers has gained prime importance, regulation of this process in cancer stem cells (CSCs) are still unrecognized. In this article, we aim to summarize the alteration of ribosome biogenesis and ribosomal proteins in CSCs. Moreover, we want to highlight the relation of ribosome biogenesis with hypoxia and drug resistance in CSCs based on the existing evidences. Lastly, this review wants to pay attention about the promising anti-cancer drugs which have potential to inhibit ribosome biogenesis in cancer cells as well as CSCs.
Assuntos
Neoplasias , Células-Tronco Neoplásicas , Proteínas Ribossômicas , Ribossomos , Humanos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Ribossomos/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Animais , Resistencia a Medicamentos Antineoplásicos , Biossíntese de ProteínasRESUMO
The ribosome utilizes hydrogen bonding between mRNA codons and aminoacyl-tRNAs to ensure rapid and accurate protein production. Chemical modification of mRNA nucleobases can adjust the strength and pattern of this hydrogen bonding to alter protein synthesis. We investigate how the N1-methylpseudouridine (m1Ψ) modification, commonly incorporated into therapeutic and vaccine mRNA sequences, influences the speed and fidelity of translation. We find that m1Ψ does not substantially change the rate constants for amino acid addition by cognate tRNAs or termination by release factors. However, we also find that m1Ψ can subtly modulate the fidelity of amino acid incorporation in a codon-position and tRNA dependent manner in vitro and in human cells. Our computational modeling shows that altered energetics of mRNA:tRNA interactions largely account for the context dependence of the low levels of miscoding we observe on Ψ and m1Ψ containing codons. The outcome of translation on modified mRNA bases is thus governed by the sequence context in which they occur.
Assuntos
Códon , Biossíntese de Proteínas , Pseudouridina , RNA Mensageiro , RNA de Transferência , Pseudouridina/metabolismo , Pseudouridina/análogos & derivados , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Humanos , Códon/genética , RNA de Transferência/metabolismo , RNA de Transferência/genética , Ribossomos/metabolismo , Ligação de Hidrogênio , Células HEK293RESUMO
Background: Ischaemic stroke is a leading cause of death and severe disability worldwide. Given the importance of protein synthesis in the inflammatory response and neuronal repair and regeneration after stroke, and that proteins are acquired by ribosomal translation of mRNA, it has been theorised that ribosome biogenesis may have an impact on promoting and facilitating recovery after stroke. However, the relationship between stroke and ribosome biogenesis has not been investigated. Methods: In the present study, a ribosome biogenesis gene signature (RSG) was developed using Cox and least absolute shrinkage and selection operator (LASSO) analysis. We classified ischaemic stroke patients into high-risk and low-risk groups using the obtained relevant genes, and further elucidated the immune infiltration of the disease using ssGSEA, which clarified the close relationship between ischaemic stroke and immune subgroups. The concentration of related proteins in the serum of stroke patients was determined by ELISA, and the patients were divided into groups to evaluate the effect of the ribosome biogenesis gene on patients. Through bioinformatics analysis, we identified potential IS-RSGs and explored future therapeutic targets, thereby facilitating the development of more effective therapeutic strategies and novel drugs against potential therapeutic targets in ischaemic stroke. Results: We obtained a set of 12 ribosome biogenesis-related genes (EXOSC5, MRPS11, MRPS7, RNASEL, RPF1, RPS28, C1QBP, GAR1, GRWD1, PELP1, UTP, ERI3), which play a key role in assessing the prognostic risk of ischaemic stroke. Importantly, risk grouping using ribosome biogenesis-related genes was also closely associated with important signaling pathways in stroke. ELISA detected the expression of C1QBP, RPS28 and RNASEL proteins in stroke patients, and the proportion of neutrophils was significantly increased in the high-risk group. Conclusions: The present study demonstrates the involvement of ribosomal biogenesis genes in the pathogenesis of ischaemic stroke, providing novel insights into the underlying pathogenic mechanisms and potential therapeutic strategies for ischaemic stroke.
Assuntos
AVC Isquêmico , Ribossomos , Humanos , AVC Isquêmico/genética , AVC Isquêmico/imunologia , Ribossomos/metabolismo , Ribossomos/genética , Masculino , Feminino , Idoso , Pessoa de Meia-Idade , Biologia Computacional/métodos , Transcriptoma , Perfilação da Expressão Gênica , Proteínas Ribossômicas/genética , BiomarcadoresRESUMO
In the complex landscape of cancer biology, the discovery of microproteins has triggered a paradigm shift, thereby, challenging the conventional conceptions of gene regulation. Though overlooked for years, these entities encoded by the small open reading frames (100-150 codons), have a significant impact on various cellular processes. As precision medicine pioneers delve deeper into the genome and proteome, microproteins have come into the limelight. Typically characterized by a single protein domain that directly binds to the target protein complex and regulates their assembly, these microproteins have been shown to play a key role in fundamental biological processes such as RNA processing, DNA repair, and metabolism regulation. Techniques for identification and characterization, such as ribosome profiling and proteogenomic approaches, have unraveled unique mechanisms by which these microproteins regulate cell signaling or pathological processes in most diseases including cancer. However, the functional relevance of these microproteins in cancer remains unclear. In this context, the current review aims to "rethink the essence of these genes" and explore "how these hidden players-microproteins orchestrate the signaling cascades of cancer, both as accelerators and brakes.".
Assuntos
Neoplasias , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Transdução de Sinais , Fases de Leitura Aberta , Animais , Ribossomos/metabolismo , Ribossomos/genética , MicropeptídeosRESUMO
Nascent chains undergo co-translational enzymatic processing as soon as their N-terminus becomes accessible at the ribosomal polypeptide tunnel exit (PTE). In eukaryotes, N-terminal methionine excision (NME) by Methionine Aminopeptidases (MAP1 and MAP2), and N-terminal acetylation (NTA) by N-Acetyl-Transferase A (NatA), is the most common combination of subsequent modifications carried out on the 80S ribosome. How these enzymatic processes are coordinated in the context of a rapidly translating ribosome has remained elusive. Here, we report two cryo-EM structures of multi-enzyme complexes assembled on vacant human 80S ribosomes, indicating two routes for NME-NTA. Both assemblies form on the 80S independent of nascent chain substrates. Irrespective of the route, NatA occupies a non-intrusive 'distal' binding site on the ribosome which does not interfere with MAP1 or MAP2 binding nor with most other ribosome-associated factors (RAFs). NatA can partake in a coordinated, dynamic assembly with MAP1 through the hydra-like chaperoning function of the abundant Nascent Polypeptide-Associated Complex (NAC). In contrast to MAP1, MAP2 completely covers the PTE and is thus incompatible with NAC and MAP1 recruitment. Together, our data provide the structural framework for the coordinated orchestration of NME and NTA in protein biogenesis.