RESUMO
Alterations in cell fate are often attributed to (epigenetic) regulation of gene expression. An emerging paradigm focuses on specialized ribosomes within a cell. However, little evidence exists for the dynamic regulation of ribosome composition and function. Here, we stimulated a chondrocytic cell line with transforming growth factor beta (TGF-ß2) and mapped changes in ribosome function, composition and ribosomal RNA (rRNA) epitranscriptomics. 35S Met/Cys incorporation was used to evaluate ribosome activity. Dual luciferase reporter assays were used to assess ribosomal modus. Ribosomal RNA expression and processing were determined by RT-qPCR, while RiboMethSeq and HydraPsiSeq were used to determine rRNA modification profiles. Label-free protein quantification of total cell lysates, isolated ribosomes and secreted proteins was done by LC-MS/MS. A three-day TGF-ß2 stimulation induced total protein synthesis in SW1353 chondrocytic cells and human articular chondrocytes. Specifically, TGF-ß2 induced cap-mediated protein synthesis, while IRES-mediated translation was not (P53 IRES) or little affected (CrPv IGR and HCV IRES). Three rRNA post-transcriptional modifications (PTMs) were affected by TGF-ß2 stimulation (18S-Gm1447 downregulated, 18S-ψ1177 and 28S-ψ4598 upregulated). Proteomic analysis of isolated ribosomes revealed increased interaction with eIF2 and tRNA ligases and decreased association of eIF4A3 and heterogeneous nuclear ribonucleoprotein (HNRNP)s. In addition, thirteen core ribosomal proteins were more present in ribosomes from TGF-ß2 stimulated cells, albeit with a modest fold change. A prolonged stimulation of chondrocytic cells with TGF-ß2 induced ribosome activity and changed the mode of translation. These functional changes could be coupled to alterations in accessory proteins in the ribosomal proteome.
Assuntos
Condrócitos , Biossíntese de Proteínas , RNA Ribossômico , Ribossomos , Fator de Crescimento Transformador beta2 , Condrócitos/metabolismo , Condrócitos/efeitos dos fármacos , Ribossomos/metabolismo , Humanos , RNA Ribossômico/metabolismo , RNA Ribossômico/genética , Fator de Crescimento Transformador beta2/metabolismo , Fator de Crescimento Transformador beta2/farmacologia , Sítios Internos de Entrada Ribossomal , Linhagem CelularRESUMO
One mechanism of particular interest to regulate mRNA fate post-transcriptionally is mRNA modification. Especially the extent of m1A mRNA methylation is highly discussed due to methodological differences. However, one single m1A site in mitochondrial ND5 mRNA was unanimously reported by different groups. ND5 is a subunit of complex I of the respiratory chain. It is considered essential for the coupling of oxidation and proton transport. Here we demonstrate that this m1A site might be involved in the pathophysiology of Alzheimer's disease (AD). One of the pathological hallmarks of this neurodegenerative disease is mitochondrial dysfunction, mainly induced by Amyloid ß (Aß). Aß mainly disturbs functions of complex I and IV of the respiratory chain. However, the molecular mechanism of complex I dysfunction is still not fully understood. We found enhanced m1A methylation of ND5 mRNA in an AD cell model as well as in AD patients. Formation of this m1A methylation is catalyzed by increased TRMT10C protein levels, leading to translation repression of ND5. As a consequence, here demonstrated for the first time, TRMT10C induced m1A methylation of ND5 mRNA leads to mitochondrial dysfunction. Our findings suggest that this newly identified mechanism might be involved in Aß-induced mitochondrial dysfunction.
Assuntos
Adenosina , Doença de Alzheimer , Peptídeos beta-Amiloides , Complexo I de Transporte de Elétrons , Mitocôndrias , RNA Mensageiro , Humanos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Mitocôndrias/metabolismo , Metilação , Complexo I de Transporte de Elétrons/metabolismo , Complexo I de Transporte de Elétrons/genética , Peptídeos beta-Amiloides/metabolismo , Masculino , Feminino , Idoso , Metiltransferases/metabolismo , Metiltransferases/genética , Idoso de 80 Anos ou mais , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genéticaRESUMO
Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N7-methylguanosine (m7G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m7G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m7G tRNA methylation in cancer cell translation control and tumour biology.
Assuntos
Carcinogênese , Neoplasias da Próstata , Masculino , Humanos , Carcinogênese/genética , Transformação Celular Neoplásica , Neoplasias da Próstata/genética , Transcrição Gênica , Processamento Pós-Transcricional do RNA , Metiltransferases/genéticaRESUMO
In plant cells, a large pool of iron (Fe) is contained in the nucleolus, as well as in chloroplasts and mitochondria. A central determinant for intracellular distribution of Fe is nicotianamine (NA) generated by NICOTIANAMINE SYNTHASE (NAS). Here, we used Arabidopsis thaliana plants with disrupted NAS genes to study the accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression. We found that nas124 triple mutant plants, which contained lower quantities of the iron ligand NA, also contained less iron in the nucleolus. This was concurrent with the expression of normally silenced rRNA genes from nucleolar organizer regions 2 (NOR2). Notably, in nas234 triple mutant plants, which also contained lower quantities of NA, nucleolar iron and rDNA expression were not affected. In contrast, in both nas124 and nas234, specific RNA modifications were differentially regulated in a genotype dependent manner. Taken together, our results highlight the impact of specific NAS activities in RNA gene expression. We discuss the interplay between NA and nucleolar iron with rDNA functional organization and RNA methylation.
Assuntos
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , DNA Ribossômico/metabolismo , Metilação , Ferro/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismoRESUMO
FUS is a multifunctional protein involved in many aspects of RNA metabolism, including transcription, splicing, translation, miRNA processing, and replication-dependent histone gene expression. In this work, we show that FUS depletion results in the differential expression of numerous small nucleolar RNAs (snoRNAs) that guide 2'-O methylation (2'-O-Me) and pseudouridylation of specific positions in ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). Using RiboMeth-seq and HydraPsiSeq for the profiling of 2'-O-Me and pseudouridylation status of rRNA species, we demonstrated considerable hypermodification at several sites in HEK293T and SH-SY5Y cells with FUS knockout (FUS KO) compared to wild-type cells. We observed a similar direction of changes in rRNA modification in differentiated SH-SY5Y cells with the FUS mutation (R495X) related to the severe disease phenotype of amyotrophic lateral sclerosis (ALS). Furthermore, the pattern of modification of some rRNA positions was correlated with the abundance of corresponding guide snoRNAs in FUS KO and FUS R495X cells. Our findings reveal a new role for FUS in modulating the modification pattern of rRNA molecules, that in turn might generate ribosome heterogeneity and constitute a fine-tuning mechanism for translation efficiency/fidelity. Therefore, we suggest that increased levels of 2'-O-Me and pseudouridylation at particular positions in rRNAs from cells with the ALS-linked FUS mutation may represent a possible new translation-related mechanism that underlies disease development and progression.
Assuntos
Esclerose Lateral Amiotrófica , Neuroblastoma , Humanos , RNA Nucleolar Pequeno/genética , Células HEK293 , RNA Ribossômico/genética , Proteína FUS de Ligação a RNA/genéticaRESUMO
FTSJ1 is a conserved human 2'-O-methyltransferase (Nm-MTase) that modifies several tRNAs at position 32 and the wobble position 34 in the anticodon loop. Its loss of function has been linked to X-linked intellectual disability (XLID), and more recently to cancers. However, the molecular mechanisms underlying these pathologies are currently unclear. Here, we report a novel FTSJ1 pathogenic variant from an X-linked intellectual disability patient. Using blood cells derived from this patient and other affected individuals carrying FTSJ1 mutations, we performed an unbiased and comprehensive RiboMethSeq analysis to map the ribose methylation on all human tRNAs and identify novel targets. In addition, we performed a transcriptome analysis in these cells and found that several genes previously associated with intellectual disability and cancers were deregulated. We also found changes in the miRNA population that suggest potential cross-regulation of some miRNAs with these key mRNA targets. Finally, we show that differentiation of FTSJ1-depleted human neural progenitor cells into neurons displays long and thin spine neurites compared with control cells. These defects are also observed in Drosophila and are associated with long-term memory deficits. Altogether, our study adds insight into FTSJ1 pathologies in humans and flies by the identification of novel FTSJ1 targets and the defect in neuron morphology.
Assuntos
Deficiência Intelectual , Ribose , Humanos , Metilação , Deficiência Intelectual/genética , Metiltransferases/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/genéticaRESUMO
Epstein-Barr virus (EBV) expresses two highly abundant noncoding RNAs called EBV-encoded RNA 1 (EBER1) and EBER2, which are preserved in all clinical isolates of EBV, thus underscoring their essential function in the viral life cycle. Recent epitranscriptomics studies have uncovered a vast array of distinct RNA modifications within cellular as well as viral noncoding RNAs that are instrumental in executing their function. Here we show that EBER2 is marked by pseudouridylation, and by using HydraPsiSeq the modification site was mapped to a single nucleotide within the 3' region of EBER2. The writer enzyme was identified to be the snoRNA-dependent pseudouridine synthase Dyskerin, which is the catalytic subunit of H/ACA small nucleolar ribonucleoprotein complexes, and is guided to EBER2 by SNORA22. Similar to other noncoding RNAs for which pseudouridylation has a positive effect on RNA stability, loss of EBER2 pseudouridylation results in a decrease in RNA levels. Furthermore, pseudouridylation of EBER2 is required for the prolific accumulation of progeny viral genomes, suggesting that this single modification in EBER2 is important for efficient viral lytic replication. Taken together, our findings add to the list of RNA modifications that are essential for noncoding RNAs to implement their physiological roles.
Assuntos
Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Humanos , Herpesvirus Humano 4/genética , RNA Viral/genética , RNA não Traduzido/genética , Estabilidade de RNA , Replicação Viral/genéticaRESUMO
Among RNA modifications, transfer of methylgroups from the typical cofactor S-adenosyl-l-methionine by methyltransferases (MTases) to RNA is by far the most common reaction. Since our last review about a decade ago, the field has witnessed the re-emergence of mRNA methylation as an important mechanism in gene regulation. Attention has then spread to many other RNA species; all being included into the newly coined concept of the "epitranscriptome." The focus moved from prokaryotes and single cell eukaryotes as model organisms to higher eukaryotes, in particular to mammals. The perception of the field has dramatically changed over the past decade. A previous lack of phenotypes in knockouts in single cell organisms has been replaced by the apparition of MTases in numerous disease models and clinical investigations. Major driving forces of the field include methylation mapping techniques, as well as the characterization of the various MTases, termed "writers." The latter term has spilled over from DNA modification in the neighboring epigenetics field, along with the designations "readers," applied to mediators of biological effects upon specific binding to a methylated RNA. Furthermore "eraser" enzymes effect the newly discovered oxidative removal of methylgroups. A sense of reversibility and dynamics has replaced the older perception of RNA modification as a concrete-cast, irreversible part of RNA maturation. A related concept concerns incompletely methylated residues, which, through permutation of each site, lead to inhomogeneous populations of numerous modivariants. This review recapitulates the major developments of the past decade outlined above, and attempts a prediction of upcoming trends. This article is categorized under: RNA Processing > RNA Editing and Modification.
Assuntos
Metiltransferases , Nucleotídeos , Animais , Epigênese Genética , Metilação , Metiltransferases/metabolismo , RNARESUMO
Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m3C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons through METTL8-mediated m3C32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain.
Assuntos
Metiltransferases/metabolismo , RNA Mitocondrial/química , RNA de Transferência/química , Animais , Anticódon , Proliferação de Células , Códon , Citoplasma , DNA Mitocondrial/metabolismo , Transporte de Elétrons , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Mitocôndrias/metabolismo , Membranas Mitocondriais , Proteínas Mitocondriais/química , Consumo de Oxigênio , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Ribossomos/metabolismo , Regulação para CimaRESUMO
Epitranscriptomic modifications of stable RNAs are dynamically regulated and specific profiles of 2'-O-methylation in rRNA have been associated with distinct cancer types. However, these observations pointed out the existence of at least two distinct groups: a rather large group with constitutive rRNA Nm residues exhibiting a stable level of methylation and a more restricted set of variable modifications, giving rise to the concept of 'specialized ribosomes'. These heterogeneous ribosomes can modulate their translational properties and be key regulatory players, depending on the physiological state of the cell. However, these conclusions were drawn from a limited set of explored human cell lines or tissues, mostly related to cancer cells of the same type. Here, we report a comprehensive analysis of human rRNA Nm modification variability observed for >15 human cell lines grown in different media and conditions. Our data demonstrate that human Nm sites can be classified into four groups, depending on their observed variability. About â of rRNA 2'-O-methylations are almost invariably modified at the same level in all tested samples (stable modifications), the second group of relatively invariant modifications (another ½ of the total) showing a slightly higher variance (low variable group) and two variable groups, showing an important heterogeneity. Mapping of these four classes on the human ribosome 3D structure shows that stably modified positions are preferentially located in the important ribosome functional sites, while variable and highly variable residues are mostly distributed to the ribosome periphery. Possible relationships of such stable and variable modifications to the ribosome functions are discussed.
Assuntos
Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , RNA Ribossômico/química , RNA Ribossômico/genética , Ribossomos/genética , Humanos , Metilação , RNA Ribossômico/metabolismo , Ribossomos/metabolismoRESUMO
[This corrects the article DOI: 10.1093/narcan/zcaa036.].
RESUMO
Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its N6,2'-O-dimethyladenosine (m6Am) demethylase activity. While m6Am is strategically located next to the m7G-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates m6Am level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear m6Am methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of m6Am modification in stem-like properties acquisition. FTO-mediated regulation of m6Am marking constitutes a reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the m6Am modification and its potential adverse consequences for colorectal cancer management.
Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Neoplasias Colorretais/metabolismo , Citoplasma/metabolismo , Desmetilação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Metiltransferases/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismoRESUMO
Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of â¼50% were detected in defined amplicons, and this threshold can be lowered to â¼10% by combination with m6A immunoprecipitation. NOseq faithfully detected known m6A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m6A candidate sites, drawn from miCLIP data with an m6A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m6A sites in defined sequences.
Assuntos
Adenosina/análogos & derivados , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/química , Análise de Sequência de RNA/métodos , Adenosina/análise , Algoritmos , Animais , Cromatografia Líquida , Desaminação , Drosophila melanogaster/genética , Células HEK293 , Células HeLa , Humanos , RNA Longo não Codificante/química , RNA Mensageiro/química , RNA Ribossômico 18S/química , Alinhamento de Sequência , Espectrometria de Massas em TandemRESUMO
Developing methods for accurate detection of RNA modifications remains a major challenge in epitranscriptomics. Next-generation sequencing-based mapping approaches have recently emerged but, often, they are not quantitative and lack specificity. Pseudouridine (ψ), produced by uridine isomerization, is one of the most abundant RNA modification. ψ mapping classically involves derivatization with soluble carbodiimide (CMCT), which is prone to variation making this approach only semi-quantitative. Here, we developed 'HydraPsiSeq', a novel quantitative ψ mapping technique relying on specific protection from hydrazine/aniline cleavage. HydraPsiSeq is quantitative because the obtained signal directly reflects pseudouridine level. Furthermore, normalization to natural unmodified RNA and/or to synthetic in vitro transcripts allows absolute measurements of modification levels. HydraPsiSeq requires minute amounts of RNA (as low as 10-50 ng), making it compatible with high-throughput profiling of diverse biological and clinical samples. Exploring the potential of HydraPsiSeq, we profiled human rRNAs, revealing strong variations in pseudouridylation levels at â¼20-25 positions out of total 104 sites. We also observed the dynamics of rRNA pseudouridylation throughout chondrogenic differentiation of human bone marrow stem cells. In conclusion, HydraPsiSeq is a robust approach for the systematic mapping and accurate quantification of pseudouridines in RNAs with applications in disease, aging, development, differentiation and/or stress response.
Assuntos
Pseudouridina/isolamento & purificação , RNA Mensageiro , RNA Ribossômico , RNA de Transferência , Análise de Sequência de RNA/métodos , Células Cultivadas , Humanos , Células-Tronco Mesenquimais , Saccharomyces cerevisiae/genéticaRESUMO
Nonsense mutations cause about 10% of genetic disease cases, and no treatments are available. Nonsense mutations can be corrected by molecules with nonsense mutation readthrough activity. An extract of the mushroom Lepista inversa has recently shown high-efficiency correction of UGA and UAA nonsense mutations. One active constituent of this extract is 2,6-diaminopurine (DAP). In Calu-6 cancer cells, in which TP53 gene has a UGA nonsense mutation, DAP treatment increases p53 level. It also decreases the growth of tumors arising from Calu-6 cells injected into immunodeficient nude mice. DAP acts by interfering with the activity of a tRNA-specific 2'-O-methyltransferase (FTSJ1) responsible for cytosine 34 modification in tRNATrp. Low-toxicity and high-efficiency UGA nonsense mutation correction make DAP a good candidate for the development of treatments for genetic diseases caused by nonsense mutations.
Assuntos
2-Aminopurina/análogos & derivados , 2-Aminopurina/farmacologia , Códon sem Sentido/efeitos dos fármacos , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Mutação/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes p53/genética , Células HEK293 , Células HeLa , Humanos , Lepisma/química , Camundongos , Camundongos Nus , RNA de Transferência/genética , tRNA Metiltransferases/metabolismoRESUMO
Reverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to train a random forest model as a machine learning regimen for prediction of modifications, we found strongly variegated success rates for the prediction of methylated purines, as exemplified with N1-methyladenosine (m1A). Among the 13 enzymes, a correlation was found between read length, misincorporation, and prediction success. Inversely, low average read length was correlated to high arrest rate and lower prediction success. The three most successful polymerases were then applied to the characterization of RT-signatures of other methylated purines. Guanosines featuring methyl groups on the Watson-Crick face were identified with high confidence, but discrimination between m1G and m22G was only partially successful. In summary, the results suggest that, given sufficient coverage and a set of specifically optimized reaction conditions for reverse transcription, all RNA modifications that impede Watson-Crick bonds can be distinguished by their RT-signature.
Assuntos
DNA Polimerase Dirigida por RNA/metabolismo , Transcrição Reversa , Adenosina/análogos & derivados , Guanosina/química , Guanosina/metabolismo , Aprendizado de Máquina , Metilação , Oligorribonucleotídeos/química , TranscriptomaRESUMO
Recent epitranscriptomics studies unravelled that ribosomal RNA (rRNA) 2'O-methylation is an additional layer of gene expression regulation highlighting the ribosome as a novel actor of translation control. However, this major finding lies on evidences coming mainly, if not exclusively, from cellular models. Using the innovative next-generation RiboMeth-seq technology, we established the first rRNA 2'O-methylation landscape in 195 primary human breast tumours. We uncovered the existence of compulsory/stable sites, which show limited inter-patient variability in their 2'O-methylation level, which map on functionally important sites of the human ribosome structure and which are surrounded by variable sites found from the second nucleotide layers. Our data demonstrate that some positions within the rRNA molecules can tolerate absence of 2'O-methylation in tumoral and healthy tissues. We also reveal that rRNA 2'O-methylation exhibits intra- and inter-patient variability in breast tumours. Its level is indeed differentially associated with breast cancer subtype and tumour grade. Altogether, our rRNA 2'O-methylation profiling of a large-scale human sample collection provides the first compelling evidence that ribosome variability occurs in humans and suggests that rRNA 2'O-methylation might represent a relevant element of tumour biology useful in clinic. This novel variability at molecular level offers an additional layer to capture the cancer heterogeneity and associates with specific features of tumour biology thus offering a novel targetable molecular signature in cancer.
Assuntos
Ácidos Nucleicos Livres , RNA Longo não Codificante , RNA Mensageiro , Archaea/genética , Ácidos Nucleicos Livres/química , Ácidos Nucleicos Livres/genética , Ácidos Nucleicos Livres/metabolismo , Humanos , Neoplasias/genética , Conformação de Ácido Nucleico , RNA Helicases/metabolismo , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Staphylococcus aureus/genéticaRESUMO
In mammals, 2'-O-methylation of RNA is a molecular signature by which the cellular innate immune system distinguishes endogenous from exogenous messenger RNA1-3. However, the molecular functions of RNA 2'-O-methylation are not well understood. Here we have purified TAR RNA-binding protein (TRBP) and its interacting partners and identified a DICER-independent TRBP complex containing FTSJ3, a putative 2'-O-methyltransferase (2'O-MTase). In vitro and ex vivo experiments show that FTSJ3 is a 2'O-MTase that is recruited to HIV RNA through TRBP. Using RiboMethSeq analysis4, we identified predominantly FTSJ3-dependent 2'-O-methylations at specific residues on the viral genome. HIV-1 viruses produced in FTSJ3 knockdown cells show reduced 2'-O-methylation and trigger expression of type 1 interferons (IFNs) in human dendritic cells through the RNA sensor MDA5. This induction of IFN-α and IFN-ß leads to a reduction in HIV expression. We have identified an unexpected mechanism used by HIV-1 to evade innate immune recognition: the recruitment of the TRBP-FTSJ3 complex to viral RNA and its 2'-O-methylation.
Assuntos
HIV-1/imunologia , HIV-1/patogenicidade , Imunidade Inata , Metiltransferases/metabolismo , RNA Helicases DEAD-box/metabolismo , Células Dendríticas/imunologia , HIV-1/genética , Células HeLa , Humanos , Interferon Tipo I/biossíntese , Interferon Tipo I/imunologia , Helicase IFIH1 Induzida por Interferon/metabolismo , Metilação , Metiltransferases/antagonistas & inibidores , Metiltransferases/deficiência , Ligação Proteica , RNA Viral/química , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismoRESUMO
The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.