RESUMEN
The X and Y chromosomes play important roles outside of human reproduction; namely, their potential contribution to human sex biases in physiology and disease. While sex biases are often thought to be an effect of hormones and environmental exposures, genes encoded on the sex chromosomes also play a role. Seventeen homologous gene pairs exist on the X and Y chromosomes whose proteins have critical functions in biology, from direct regulation of transcription and translation to intercellular signaling and formation of extracellular structures. In this review, we cover the current understanding of several of these sex chromosome-encoded protein homologs that are involved in transcription and chromatin regulation: SRY/SOX3, ZFX/ZFY, KDM5C/KDM5D, UTX/UTY, and TBL1X/TBL1Y. Their mechanisms of gene regulation are discussed, including any redundancies or divergent roles of the X- and Y-chromosome homologs. Additionally, we discuss associated diseases related to these proteins and any sex biases that exist therein in an effort to drive further research into how these pairs contribute to sexually dimorphic gene regulation in health and disease.
RESUMEN
As phase separation is found in an increasing variety of biological contexts, additional challenges have arisen in understanding the underlying principles of condensate formation and function. We spoke with researchers across disciplines about their views on the ever-changing landscape of biomolecular condensates.
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Condensados Biomoleculares , Investigadores , Humanos , BiologíaRESUMEN
Several rRNA-modifying enzymes install rRNA modifications while participating in ribosome assembly. Here, we show that 18S rRNA methyltransferase DIMT1 is essential for acute myeloid leukemia (AML) proliferation through a noncatalytic function. We reveal that targeting a positively charged cleft of DIMT1, remote from the catalytic site, weakens the binding of DIMT1 to rRNA and mislocalizes DIMT1 to the nucleoplasm, in contrast to the primarily nucleolar localization of wild-type DIMT1. Mechanistically, rRNA binding is required for DIMT1 to undergo liquid-liquid phase separation, which explains the distinct nucleoplasm localization of the rRNA binding-deficient DIMT1. Re-expression of wild-type or a catalytically inactive mutant E85A, but not the rRNA binding-deficient DIMT1, supports AML cell proliferation. This study provides a new strategy to target DIMT1-regulated AML proliferation via targeting this essential noncatalytic region.
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Leucemia Mieloide Aguda , Metiltransferasas , Humanos , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Leucemia Mieloide Aguda/genética , Metiltransferasas/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico 18S/metabolismoRESUMEN
Sex differences are pervasive in human health and disease. One major key to sex-biased differences lies in the sex chromosomes. Although the functions of the X chromosome proteins are well appreciated, how they compare with their Y chromosome homologs remains elusive. Herein, using ensemble and single-molecule techniques, we report that the sex chromosome-encoded RNA helicases DDX3X and DDX3Y are distinct in their propensities for liquid-liquid phase separation (LLPS), dissolution, and translation repression. We demonstrate that the N-terminal intrinsically disordered region of DDX3Y more strongly promotes LLPS than the corresponding region of DDX3X and that the weaker ATPase activity of DDX3Y, compared with DDX3X, contributes to the slower disassembly dynamics of DDX3Y-positive condensates. Interestingly, DDX3Y-dependent LLPS represses mRNA translation and enhances aggregation of FUS more strongly than DDX3X-dependent LLPS. Our study provides a platform for future comparisons of sex chromosome-encoded protein homologs, providing insights into sex differences in RNA metabolism and human disease.
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ARN Helicasas DEAD-box , ARN Helicasas , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Femenino , Humanos , Masculino , Antígenos de Histocompatibilidad Menor/metabolismo , Biosíntesis de Proteínas , Proteínas/metabolismo , ARN/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismoRESUMEN
Arthropod-borne viruses, including the alphavirus chikungunya virus (CHIKV), cause acute disease in millions of people and utilize potent mechanisms to antagonize and circumvent innate immune pathways including the type I interferon (IFN) pathway. In response, hosts have evolved antiviral counterdefense strategies that remain incompletely understood. Recent studies have found that long noncoding RNAs (lncRNAs) regulate classical innate immune pathways; how lncRNAs contribute to additional antiviral counterdefenses remains unclear. Using high-throughput genetic screening, we identified a cytoplasmic antiviral lncRNA that we named antiviral lncRNA prohibiting human alphaviruses (ALPHA), which is transcriptionally induced by alphaviruses and functions independently of IFN to inhibit the replication of CHIKV and its closest relative, O'nyong'nyong virus (ONNV), but not other viruses. Furthermore, we showed that ALPHA interacts with CHIKV genomic RNA and restrains viral RNA replication. Together, our findings reveal that ALPHA and potentially other lncRNAs can mediate non-canonical antiviral immune responses against specific viruses.
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Virus Chikungunya , Interferón Tipo I , ARN Largo no Codificante , Antivirales/farmacología , Virus Chikungunya/genética , Humanos , Inmunidad Innata/genética , Interferón Tipo I/genética , ARN Largo no Codificante/genética , ARN Viral/genética , Replicación Viral/genéticaRESUMEN
RNA binding proteins (RBPs) are important players in RNA metabolism and gene regulation. In this issue of Genes & Development, Flamand and colleagues (pp. 1002-1015) developed a new method (TRIBE-STAMP) that detects binding events by two distinct RBPs on single mRNA molecules, which they first applied to the YTHDF family of N 6-methyladenosine (m6A) reader proteins. The investigators show that these RBPs largely share a common pool of bound transcripts and that an individual mRNA may be bound by multiple YTHDF proteins throughout its lifetime. This single-molecule technique is an exciting new method to study potential synergy and/or antagonism between different RBPs.
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Regulación de la Expresión Génica , Proteínas de Unión al ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARN Mensajero/metabolismo , ARNRESUMEN
Enzyme-mediated chemical modifications of nucleic acids are indispensable regulators of gene expression. Our understanding of the biochemistry and biological significance of these modifications has largely been driven by an ever-evolving landscape of technologies that enable accurate detection, mapping, and manipulation of these marks. Here we provide a summary of recent technical advances in the study of nucleic acid modifications with a focus on techniques that allow accurate detection and mapping of these modifications. For each modification discussed (N6-methyladenosine, 5-methylcytidine, inosine, pseudouridine, and N4-acetylcytidine), we begin by introducing the "gold standard" technique for its mapping and detection, followed by a discussion of techniques developed to address any shortcomings of the gold standard. By highlighting the commonalities and differences of these techniques, we hope to provide a perspective on the current state of the field and to lay out a guideline for development of future technologies.
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Metilación de ADN , ADN/metabolismo , Técnicas Genéticas , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , ARN/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Animales , Citidina/análogos & derivados , Citidina/metabolismo , ADN/genética , Epigénesis Genética , Humanos , Inosina/metabolismo , Seudouridina/metabolismo , ARN/genética , ARN Mensajero/genéticaRESUMEN
Gene expression regulation is a critical process throughout the body, especially in the nervous system. One mechanism by which biological systems regulate gene expression is via enzyme-mediated RNA modifications, also known as epitranscriptomic regulation. RNA modifications, which have been found on nearly all RNA species across all domains of life, are chemically diverse covalent modifications of RNA nucleotides and represent a robust and rapid mechanism for the regulation of gene expression. Although numerous studies have been conducted regarding the impact that single modifications in single RNA molecules have on gene expression, emerging evidence highlights potential crosstalk between and coordination of modifications across RNA species. These potential coordination axes of RNA modifications have emerged as a new direction in the field of epitranscriptomic research. In this review, we will highlight several examples of gene regulation via RNA modification in the nervous system, followed by a summary of the current state of the field of RNA modification coordination axes. In doing so, we aim to inspire the field to gain a deeper understanding of the roles of RNA modifications and coordination of these modifications in the nervous system.
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Regulación de la Expresión Génica , ARN , ARN/genética , Encéfalo/metabolismo , Epigénesis GenéticaRESUMEN
The synthesis of quinolinic acid from tryptophan is a critical step in the de novo biosynthesis of nicotinamide adenine dinucleotide (NAD+) in mammals. Herein, the nonheme iron-based 3-hydroxyanthranilate-3,4-dioxygenase responsible for quinolinic acid production was studied by performing time-resolved in crystallo reactions monitored by UV-vis microspectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and X-ray crystallography. Seven catalytic intermediates were kinetically and structurally resolved in the crystalline state, and each accompanies protein conformational changes at the active site. Among them, a monooxygenated, seven-membered lactone intermediate as a monodentate ligand of the iron center at 1.59-Å resolution was captured, which presumably corresponds to a substrate-based radical species observed by EPR using a slurry of small-sized single crystals. Other structural snapshots determined at around 2.0-Å resolution include monodentate and subsequently bidentate coordinated substrate, superoxo, alkylperoxo, and two metal-bound enol tautomers of the unstable dioxygenase product. These results reveal a detailed stepwise O-atom transfer dioxygenase mechanism along with potential isomerization activity that fine-tunes product profiling and affects the production of quinolinic acid at a junction of the metabolic pathway.
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3-Hidroxiantranilato 3,4-Dioxigenasa/química , Proteínas Bacterianas/química , Cupriavidus/enzimología , 3-Hidroxiantranilato 3,4-Dioxigenasa/genética , 3-Hidroxiantranilato 3,4-Dioxigenasa/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Cupriavidus/química , Cupriavidus/genética , Cinética , Lactonas/química , Lactonas/metabolismo , Modelos Moleculares , Especificidad por SustratoRESUMEN
The posttranscriptional modification of messenger RNA (mRNA) and transfer RNA (tRNA) provides an additional layer of regulatory complexity during gene expression. Here, we show that a tRNA methyltransferase, TRMT10A, interacts with an mRNA demethylase FTO (ALKBH9), both in vitro and inside cells. TRMT10A installs N1-methylguanosine (m1G) in tRNA, and FTO performs demethylation on N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) in mRNA. We show that TRMT10A ablation not only leads to decreased m1G in tRNA but also significantly increases m6A levels in mRNA. Cross-linking and immunoprecipitation, followed by high-throughput sequencing results show that TRMT10A shares a significant overlap of associated mRNAs with FTO, and these mRNAs have accelerated decay rates potentially through the regulation by a specific m6A reader, YTHDF2. Furthermore, transcripts with increased m6A upon TRMT10A ablation contain an overrepresentation of m1G9-containing tRNAs codons read by tRNAGln(TTG), tRNAArg(CCG), and tRNAThr(CGT) These findings collectively reveal the presence of coordinated mRNA and tRNA methylations and demonstrate a mechanism for regulating gene expression through the interactions between mRNA and tRNA modifying enzymes.
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Adenosina/genética , Metiltransferasas/genética , ARN Mensajero/genética , ARN de Transferencia/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Regulación de la Expresión Génica/genética , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Metilación , Proteínas de Unión al ARN/genética , ARNt Metiltransferasas/genéticaRESUMEN
Dimethyladenosine transferase 1 (DIMT1) is an evolutionarily conserved RNA N6,6-dimethyladenosine (m26,6A) methyltransferase. DIMT1 plays an important role in ribosome biogenesis, and the catalytic activity of DIMT1 is indispensable for cell viability and protein synthesis. A few RNA-modifying enzymes can install the same modification in multiple RNA species. However, whether DIMT1 can work on RNA species other than 18S rRNA is unclear. Here, we describe that DIMT1 generates m26,6A not only in 18S rRNA but also in small RNAs. In addition, m26,6A in small RNAs were significantly decreased in cells expressing catalytically inactive DIMT1 variants (E85A or NLPY variants) compared with cells expressing wildtype DIMT1. Both E85A and NLPY DIMT1 variant cells present decreased protein synthesis and cell viability. Furthermore, we observed that DIMT1 is highly expressed in human cancers, including acute myeloid leukemia. Our data suggest that downregulation of DIMT1 in acute myeloid leukemia cells leads to a decreased m26,6A level in small RNAs. Together, these data suggest that DIMT1 not only installs m26,6A in 18S rRNA but also generates m26,6A-containing small RNAs, both of which potentially contribute to the impact of DIMT1 on cell viability and gene expression.
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Leucemia Mieloide Aguda/enzimología , Metiltransferasas/metabolismo , Proteínas de Neoplasias/metabolismo , Procesamiento Postranscripcional del ARN , ARN Neoplásico/metabolismo , Sustitución de Aminoácidos , Células HEK293 , Humanos , Leucemia Mieloide Aguda/genética , Metilación , Metiltransferasas/genética , Mutación Missense , Proteínas de Neoplasias/genética , ARN Neoplásico/genéticaRESUMEN
Oxidation of 5-methylcytosine (5mC) in DNA by the ten-eleven translocation (TET) family of enzymes is indispensable for gene regulation in mammals. More recently, evidence has emerged to support a biological function for TET-mediated m5C oxidation in messenger RNA. Here, we describe a previously uncharacterized role of TET-mediated m5C oxidation in transfer RNA (tRNA). We found that the TET-mediated oxidation product 5-hydroxylmethylcytosine (hm5C) is specifically enriched in tRNA inside cells and that the oxidation activity of TET2 on m5C in tRNAs can be readily observed in vitro. We further observed that hm5C levels in tRNA were significantly decreased in Tet2 KO mouse embryonic stem cells (mESCs) in comparison with wild-type mESCs. Reciprocally, induced expression of the catalytic domain of TET2 led to an obvious increase in hm5C and a decrease in m5C in tRNAs relative to uninduced cells. Strikingly, we also show that TET2-mediated m5C oxidation in tRNA promotes translation in vitro. These results suggest TET2 may influence translation through impacting tRNA methylation and reveal an unexpected role for TET enzymes in regulating multiple nodes of the central dogma.
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5-Metilcitosina/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , ARN de Transferencia/metabolismo , 5-Metilcitosina/química , Animales , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Dioxigenasas , Células Madre Embrionarias/metabolismo , Ratones , Ratones Noqueados , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas/química , ARN de Transferencia/químicaRESUMEN
rRNA-modifying enzymes participate in ribosome assembly. However, whether the catalytic activities of these enzymes are important for the ribosome assembly and other cellular processes is not fully understood. Here, we report the crystal structure of WT human dimethyladenosine transferase 1 (DIMT1), an 18S rRNA N6,6-dimethyladenosine (m26,6A) methyltransferase, and results obtained with a catalytically inactive DIMT1 variant. We found that DIMT1+/- heterozygous HEK 293T cells have a significantly decreased 40S fraction and reduced protein synthesis but no major changes in m26,6A levels in 18S rRNA. Expression of a catalytically inactive variant, DIMT1-E85A, in WT and DIMT1+/- cells significantly decreased m26,6A levels in 18S rRNA, indicating a dominant-negative effect of this variant on m26,6A levels. However, expression of the DIMT1-E85A variant restored the defects in 40S levels. Of note, unlike WT DIMT1, DIMT1-E85A could not revert the defects in protein translation. We found that the differences between this variant and the WT enzyme extended to translation fidelity and gene expression patterns in DNA damage response pathways. These results suggest that the catalytic activity of DIMT1 is involved in protein translation and that the overall protein scaffold of DIMT1, regardless of the catalytic activity on m26,6A in 18S rRNA, is essential for 40S assembly.
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Metiltransferasas/química , Biosíntesis de Proteínas , ARN Ribosómico 18S/química , Sustitución de Aminoácidos , Catálisis , Cristalografía por Rayos X , Células HEK293 , Humanos , Metiltransferasas/genética , Metiltransferasas/metabolismo , Mutación Missense , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/química , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismoRESUMEN
Nucleic acid modifications in DNA and RNA ubiquitously exist among all the three kingdoms of life. This trait significantly broadens the genome diversity and works as an important means of gene transcription regulation. Although mammalian systems have limited types of DNA modifications, over 150 different RNA modification types have been identified, with a wide variety of chemical diversities. Most modifications occur on transfer RNA and ribosomal RNA, however many of the modifications also occur on other types of RNA species including mammalian mRNA and small nuclear RNA, where they are essential for many biological roles, including developmental processes and stem cell differentiation. These post-transcriptional modifications are enzymatically installed and removed in a site-specific manner by writer and eraser proteins respectively, while reader proteins can interpret modifications and transduce the signal for downstream functions. Dysregulation of mRNA modifications manifests as disease states, including multiple types of human cancer. In this review, we will introduce the chemical features and biological functions of these modifications in the coding and non-coding RNA species.
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Diferenciación Celular/fisiología , Procesamiento Postranscripcional del ARN/fisiología , ARN Mensajero/metabolismo , ARN Ribosómico/metabolismo , ARN de Transferencia/metabolismo , Células Madre/metabolismo , Animales , Humanos , Células Madre/citologíaRESUMEN
The complexity of biological sex differences is markedly evident in human physiology and pathology. Although many of these differences can be ascribed to the expression of sex hormones, another contributor to sex differences lies in the sex chromosomes beyond their role in sex determination. Although largely nonhomologous, the human sex chromosomes express seventeen pairs of homologous genes, referred to as the 'X-Y pairs.' The X chromosome-encoded homologs of these Y-encoded proteins are crucial players in several cellular processes, and their dysregulation frequently results in disease development. Many diseases related to these X-encoded homologs present with sex-biased incidence or severity. By contrast, comparatively little is known about the differential functions of the Y-linked homologs. Here, we summarize and discuss the current understanding of five of these X-Y paired proteins, with recent evidence of differential functions and of having a potential link to sex biases in disease, highlighting how amino acid-level sequence differences may differentiate their functions and contribute to sex biases in human disease.
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DEAD-box helicases, which are crucial for many aspects of RNA metabolism, often contain intrinsically disordered regions (IDRs), whose functions remain unclear. Using multiparameter confocal microscopy, we reveal that sex chromosome-encoded homologous RNA helicases, DDX3X and DDX3Y, form nano-sized RNA-protein clusters (RPCs) that foster their catalytic activities in vitro and in cells. The IDRs are critical for the formation of these RPCs. A thorough analysis of the catalytic cycle of DDX3X and DDX3Y by ensemble biochemistry and single molecule photon bursts in the confocal microscope showed that RNA release is a major step that differentiates the unwinding activities of DDX3X and DDX3Y. Our findings provide new insights that the nano-sized helicase RPCs may be the normal state of these helicases under non-stressed conditions that promote their RNA unwinding and act as nucleation points for liquid-liquid phase separation under stress. This mechanism may apply broadly among other members of the DEAD-box helicase family.
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Mutations in the RNA helicase DDX3X, implicated in various cancers and neurodevelopmental disorders, often impair RNA unwinding and translation. However, the mechanisms underlying this impairment and the differential interactions of DDX3X mutants with wild-type (WT) X-linked DDX3X and Y-linked homolog DDX3Y remain elusive. This study reveals that specific DDX3X mutants more frequently found in disease form distinct hollow condensates in cells. Using a combined structural, biochemical, and single-molecule microscopy study, we show that reduced ATPase and RNA release activities contribute to condensate formation and the catalytic deficits result from inhibiting the catalytic cycle at multiple steps. Proteomic investigations further demonstrate that these hollow condensates sequester WT DDX3X/DDX3Y and other proteins crucial for diverse signaling pathways. WT DDX3X enhances the dynamics of heterogeneous mutant/WT hollow condensates more effectively than DDX3Y. These findings offer valuable insights into the catalytic defects of specific DDX3X mutants and their differential interactions with wild-type DDX3X and DDX3Y, potentially explaining sex biases in disease.
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N6-methyladenosine (m6A) is the most abundant modification on messenger RNAs (mRNAs) and is catalyzed by methyltransferase-like protein 3 (Mettl3). To understand the role of m6A in a self-renewing somatic tissue, we deleted Mettl3 in epidermal progenitors in vivo. Mice lacking Mettl3 demonstrate marked features of dysfunctional development and self-renewal, including a loss of hair follicle morphogenesis and impaired cell adhesion and polarity associated with oral ulcerations. We show that Mettl3 promotes the m6A-mediated degradation of mRNAs encoding critical histone modifying enzymes. Depletion of Mettl3 results in the loss of m6A on these mRNAs and increases their expression and associated modifications, resulting in widespread gene expression abnormalities that mirror the gross phenotypic abnormalities. Collectively, these results have identified an additional layer of gene regulation within epithelial tissues, revealing an essential role for m6A in the regulation of chromatin modifiers, and underscoring a critical role for Mettl3-catalyzed m6A in proper epithelial development and self-renewal.
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Histonas , Metiltransferasas , Animales , Ratones , Metiltransferasas/genética , Adenosina , Adhesión Celular , ARN Mensajero , CatálisisRESUMEN
N6-methyladenosine (m6A), the most prevalent internal modification on eukaryotic mRNA, plays an essential role in various stress responses. The brain is uniquely vulnerable to cellular stress, thus defining how m6A sculpts the brain's susceptibility may provide insight to brain aging and disease-related stress. Here we investigate the impact of m6A mRNA methylation in the adult Drosophila brain with stress. We show that m6A is enriched in the adult brain and increases with heat stress. Through m6A-immunoprecipitation sequencing, we show 5'UTR Mettl3-dependent m6A is enriched in transcripts of neuronal processes and signaling pathways that increase upon stress. Mettl3 knockdown results in increased levels of m6A targets and confers resilience to stress. We find loss of Mettl3 results in decreased levels of nuclear m6A reader Ythdc1, and knockdown of Ythdc1 also leads to stress resilience. Overall, our data suggest that m6A modification in Drosophila dampens the brain's biological response to stress.
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Adenosina , Drosophila , Adenosina/metabolismo , Animales , Encéfalo/metabolismo , Drosophila/genética , Drosophila/metabolismo , Metilación , ARN Mensajero/metabolismoRESUMEN
The insulin responsive Akt and FoxO1 signaling axis is a key regulator of the hepatic transcriptional response to nutrient intake. Here, we used global run-on sequencing (GRO-seq) to measure the nascent transcriptional response to fasting and refeeding as well as define the specific role of hepatic Akt and FoxO1 signaling in mediating this response. We identified 599 feeding-regulated transcripts, as well as over 6,000 eRNAs, and mapped their dependency on Akt and FoxO1 signaling. Further, we identified several feeding-regulated lncRNAs, including the lncRNA Gm11967, whose expression was dependent upon the liver Akt-FoxO1 axis. Restoring Gm11967 expression in mice lacking liver Akt improved insulin sensitivity and induced glucokinase protein expression, indicating that Akt-dependent control of Gm11967 contributes to the translational control of glucokinase. More broadly, we have generated a unique genome-wide dataset that defines the feeding and Akt/FoxO1-dependent transcriptional changes in response to nutrient availability.