RESUMEN
Tumor-suppressor let-7 pre-microRNAs (miRNAs) are regulated by terminal uridylyltransferases TUT7 and TUT4 that either promote let-7 maturation by adding a single uridine nucleotide to the pre-miRNA 3' end or mark them for degradation by the addition of multiple uridines. Oligo-uridylation is increased in cells by enhanced TUT7/4 expression and especially by the RNA-binding pluripotency factor LIN28A. Using cryogenic electron microscopy, we captured high-resolution structures of active forms of TUT7 alone, of TUT7 plus pre-miRNA and of both TUT7 and TUT4 bound with pre-miRNA and LIN28A. Our structures reveal that pre-miRNAs engage the enzymes in fundamentally different ways depending on the presence of LIN28A, which clamps them onto the TUTs to enable processive 3' oligo-uridylation. This study reveals the molecular basis for mono- versus oligo-uridylation by TUT7/4, as determined by the presence of LIN28A, and thus their mechanism of action in the regulation of cell fate and in cancer.
Asunto(s)
Microscopía por Crioelectrón , MicroARNs , Proteínas de Unión al ARN , Humanos , MicroARNs/metabolismo , MicroARNs/genética , MicroARNs/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/química , Modelos Moleculares , ARN Nucleotidiltransferasas/metabolismo , ARN Nucleotidiltransferasas/química , ARN Nucleotidiltransferasas/genética , Precursores del ARN/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/química , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/química , Conformación ProteicaRESUMEN
The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.
Asunto(s)
Citoplasma , Homeostasis , ARN Mensajero , Gránulos de Estrés , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Gránulos de Estrés/metabolismo , Citoplasma/metabolismo , Caperuzas de ARN/metabolismo , Arsenitos/farmacología , Estrés Oxidativo , Transporte Activo de Núcleo Celular , ARN Nucleotidiltransferasas/metabolismo , ARN Nucleotidiltransferasas/genética , Compuestos de Sodio/farmacología , Proteína Exportina 1 , Carioferinas/metabolismo , Carioferinas/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Gránulos Citoplasmáticos/metabolismo , Estabilidad del ARN , Núcleo Celular/metabolismo , Línea Celular Tumoral , NucleotidiltransferasasRESUMEN
The majority of genic transcription is intronic. Introns are removed by splicing as branched lariat RNAs which require rapid recycling. The branch site is recognized during splicing catalysis and later debranched by Dbr1 in the rate-limiting step of lariat turnover. Through generation of a viable DBR1 knockout cell line, we find the predominantly nuclear Dbr1 enzyme to encode the sole debranching activity in human cells. Dbr1 preferentially debranches substrates that contain canonical U2 binding motifs, suggesting that branchsites discovered through sequencing do not necessarily represent those favored by the spliceosome. We find that Dbr1 also exhibits specificity for particular 5' splice site sequences. We identify Dbr1 interactors through co-immunoprecipitation mass spectrometry. We present a mechanistic model for Dbr1 recruitment to the branchpoint through the intron-binding protein AQR. In addition to a 20-fold increase in lariats, Dbr1 depletion increases exon skipping. Using ADAR fusions to timestamp lariats, we demonstrate a defect in spliceosome recycling. In the absence of Dbr1, spliceosomal components remain associated with the lariat for a longer period of time. As splicing is co-transcriptional, slower recycling increases the likelihood that downstream exons will be available for exon skipping.
Asunto(s)
Intrones , Empalme del ARN , Empalmosomas , Humanos , Intrones/genética , Empalmosomas/metabolismo , Células HEK293 , ARN Nucleotidiltransferasas/metabolismo , ARN Nucleotidiltransferasas/genética , Exones/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Células HeLa , Sitios de Empalme de ARNRESUMEN
Mycobacterium tuberculosis transfer RNA (tRNA) terminal nucleotidyltransferase toxin, MenT3, incorporates nucleotides at the 3'-CCA end of tRNAs, blocking their aminoacylation and inhibiting protein synthesis. Here, we show that MenT3 most effectively adds CMPs to the 3'-CCA end of tRNA. The crystal structure of MenT3 in complex with CTP reveals a CTP-specific nucleotide-binding pocket. The 4-NH2 and the N3 and O2 atoms of cytosine in CTP form hydrogen bonds with the main-chain carbonyl oxygen of P120 and the side chain of R238, respectively. MenT3 expression in Escherichia coli selectively reduces the levels of seryl-tRNASers, indicating specific inactivation of tRNASers by MenT3. Consistently, MenT3 incorporates CMPs into tRNASer most efficiently, among the tested E. coli tRNA species. The longer variable loop unique to class II tRNASers is crucial for efficient CMP incorporation into tRNASer by MenT3. Replacing the variable loop of E. coli tRNAAla with the longer variable loop of M. tuberculosis tRNASer enables MenT3 to incorporate CMPs into the chimeric tRNAAla. The N-terminal positively charged region of MenT3 is required for CMP incorporation into tRNASer. A docking model of tRNA onto MenT3 suggests that an interaction between the N-terminal region and the longer variable loop of tRNASer facilitates tRNA substrate selection.
Asunto(s)
Mycobacterium tuberculosis , ARN de Transferencia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/enzimología , Especificidad por Sustrato , ARN de Transferencia/metabolismo , ARN de Transferencia/química , Escherichia coli/genética , Escherichia coli/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Modelos Moleculares , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Citidina/química , Citidina/metabolismo , Sitios de Unión , Cristalografía por Rayos X , ARN Nucleotidiltransferasas/metabolismo , ARN Nucleotidiltransferasas/química , ARN Nucleotidiltransferasas/genéticaRESUMEN
In reference to gene annotation, more than half of the tRNA species synthesized by Mycobacterium tuberculosis require the enzymatic addition of the cytosine-cytosine-adenine (CCA) tail, which is indispensable for amino acid charging and tRNA functionality. It makes the mycobacterial CCA-adding enzyme essential for survival of the bacterium and a potential target for novel pipelines in drug discovery avenues. Here, we described the rv3907c gene product, originally annotated as poly(A)polymerase (rv3907c, PcnA) as a functional CCA-adding enzyme (CCAMtb) essential for viability of M. tuberculosis. The depletion of the enzyme affected tRNAs maturation, inhibited bacilli growth, and resulted in abundant accumulation of polyadenylated RNAs. We determined the enzymatic activities displayed by the mycobacterial CCAMtb in vitro and studied the effects of inhibiting of its transcription in bacterial cells. We are the first to properly confirm the existence of RNA polyadenylation in mycobacteria, a previously controversial phenomenon, which we found promoted upon CCA-adding enzyme downexpression.
Asunto(s)
Mycobacterium tuberculosis , Poliadenilación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Adenina , Citosina , Conformación de Ácido Nucleico , ARN Nucleotidiltransferasas/genética , ARN de Transferencia/metabolismoRESUMEN
In eukaryotic cells, the introns are excised from pre-mRNA by the spliceosome. These introns typically have a lariat configuration due to the 2'-5' phosphodiester bond between an internal branched residue and the 5' terminus of the RNA. The only enzyme known to selectively hydrolyze the 2'-5' linkage of these lariats is the RNA lariat debranching enzyme Dbr1. In humans, Dbr1 is involved in processes such as class-switch recombination of immunoglobulin genes, and its dysfunction is implicated in viral encephalitis, HIV, ALS, and cancer. However, mechanistic details of precisely how Dbr1 affects these processes are missing. Here we show that human Dbr1 contains a disordered C-terminal domain through sequence analysis and nuclear magnetic resonance. This domain stabilizes Dbr1 in vitro by reducing aggregation but is dispensable for debranching activity. We establish that Dbr1 requires Fe2+ for efficient catalysis and demonstrate that the noncatalytic protein Drn1 and the uncharacterized protein trichothiodystrophy nonphotosensitive 1 directly bind to Dbr1. We demonstrate addition of trichothiodystrophy nonphotosensitive 1 to in vitro debranching reactions increases the catalytic efficiency of human Dbr1 19-fold but has no effect on the activity of Dbr1 from the amoeba Entamoeba histolytica, which lacks a disordered C-terminal domain. Finally, we systematically examine how the identity of the branchpoint nucleotide affects debranching rates. These findings describe new aspects of Dbr1 function in humans and further clarify how Dbr1 contributes to human health and disease.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , ARN Nucleotidiltransferasas , Humanos , Intrones , ARN Nucleotidiltransferasas/genética , ARN Nucleotidiltransferasas/metabolismo , Empalme del ARN , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Activación Enzimática/genética , Dominios Proteicos , Unión Proteica , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Entamoeba histolytica/enzimología , Entamoeba histolytica/genética , Metales Pesados/metabolismoRESUMEN
Biomolecular condensates have been shown to interact in vivo, yet it is unclear whether these interactions are functionally meaningful. Here, we demonstrate that cooperativity between two distinct condensates-germ granules and P bodies-is required for transgenerational gene silencing in C. elegans. We find that P bodies form a coating around perinuclear germ granules and that P body components CGH-1/DDX6 and CAR-1/LSM14 are required for germ granules to organize into sub-compartments and concentrate small RNA silencing factors. Functionally, while the P body mutant cgh-1 is competent to initially trigger gene silencing, it is unable to propagate the silencing to subsequent generations. Mechanistically, we trace this loss of transgenerational silencing to defects in amplifying secondary small RNAs and the stability of WAGO-4 Argonaute, both known carriers of gene silencing memories. Together, these data reveal that cooperation between condensates results in an emergent capability of germ cells to establish heritable memory.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , ARN Interferente Pequeño/genética , Silenciador del Gen , Interferencia de ARN , Células Germinativas/metabolismo , ARN Nucleotidiltransferasas/genéticaRESUMEN
The pre-mRNA life cycle requires intron processing; yet, how intron-processing defects influence splicing and gene expression is unclear. Here, we find that TTDN1/MPLKIP, which is encoded by a gene implicated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to spliceosomal function. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, whereas its N-terminal intrinsically disordered region (IDR) binds the intron-binding complex (IBC). TTDN1 loss, or a mutated IDR, causes significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells. A Ttdn1-deficient mouse model recapitulates intron-processing defects and certain neurodevelopmental phenotypes seen in NP-TTD. Fusing DBR1 to the TTDN1 IDR is sufficient to recruit DBR1 to the IBC and circumvents the functional requirement for TTDN1. Collectively, our findings link RNA lariat processing with splicing outcomes by revealing the molecular function of TTDN1.
Asunto(s)
Síndromes de Tricotiodistrofia , Animales , Ratones , Intrones/genética , Síndromes de Tricotiodistrofia/genética , ARN Nucleotidiltransferasas/genética , Empalme del ARNRESUMEN
Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. Here, we show that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing.
Asunto(s)
Insuficiencia de Crecimiento , ARN Nucleotidiltransferasas , Animales , Humanos , Ratones , Ratones Noqueados , Debilidad Muscular/genética , Músculos , ARN Nucleotidiltransferasas/química , ARN Nucleotidiltransferasas/genética , Pez CebraRESUMEN
The RNA lariat debranching enzyme is the sole enzyme responsible for hydrolyzing the 2'-5' phosphodiester bond in RNA lariats produced by the spliceosome. Here, we test the ability of Dbr1 to hydrolyze branched RNAs (bRNAs) that contain a 2'-5'-phosphorothioate linkage, a modification commonly used to resist degradation. We attempted to cocrystallize a phosphorothioate-branched RNA (PS-bRNA) with wild-type Entamoeba histolytica Dbr1 (EhDbr1) but observed in-crystal hydrolysis of the phosphorothioate bond. The crystal structure revealed EhDbr1 in a product-bound state, with the hydrolyzed 2'-5' fragment of the PS-bRNA mimicking the binding mode of the native bRNA substrate. These findings suggest that product inhibition may contribute to the kinetic mechanism of Dbr1. We show that Dbr1 enzymes cleave phosphorothioate linkages at rates â¼10,000-fold more slowly than native phosphate linkages. This new product-bound crystal structure offers atomic details, which can aid inhibitor design. Dbr1 inhibitors could be therapeutic or investigative compounds for human diseases such as human immunodeficiency virus (HIV), amyotrophic lateral sclerosis (ALS), cancer, and viral encephalitis.
Asunto(s)
ARN Nucleotidiltransferasas , ARN , Humanos , ARN/química , ARN Nucleotidiltransferasas/genética , ARN Nucleotidiltransferasas/metabolismo , Empalme del ARN , Fosfatos/metabolismoRESUMEN
The terminal nucleotidyltransferases TUT4 and TUT7 (TUT4/7) regulate miRNA and mRNA stability by 3' end uridylation. In humans, TUT4/7 polyuridylates both mRNA and pre-miRNA, leading to degradation by the U-specific exonuclease DIS3L2. We investigate the role of uridylation-dependent decay in maintaining the transcriptome by transcriptionally profiling TUT4/7 deleted cells. We found that while the disruption of TUT4/7 expression increases the abundance of a variety of miRNAs, the let-7 family of miRNAs is the most impacted. Eight let-7 family miRNAs were increased in abundance in TUT4/7 deleted cells, and many let-7 mRNA targets are decreased in abundance. The mRNAs with increased abundance in the deletion strain are potential direct targets of TUT4/7, with transcripts coding for proteins involved in cellular stress response, rRNA processing, ribonucleoprotein complex biogenesis, cell-cell signaling, and regulation of metabolic processes most affected in the TUT4/7 knockout cells. We found that TUT4/7 indirectly control oncogenic signaling via the miRNA let-7a, which regulates AKT phosphorylation status. Finally, we find that, similar to fission yeast, the disruption of uridylation-dependent decay leads to major rearrangements of the transcriptome and reduces cell proliferation and adhesion.
Asunto(s)
MicroARNs , ARN Nucleotidiltransferasas , Estabilidad del ARN , Humanos , MicroARNs/genética , MicroARNs/metabolismo , ARN Nucleotidiltransferasas/genética , ARN Nucleotidiltransferasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
TENTs generate miRNA isoforms by 3' tailing. However, little is known about how tailing regulates miRNA function. Here, we generate isogenic HEK293T cell lines in which TENT2, TUT4 and TUT7 are knocked out individually or in combination. Together with rescue experiments, we characterize TENT-specific effects by deep sequencing, Northern blot and in vitro assays. We find that 3' tailing is not random but highly specific. In addition to its known adenylation, TENT2 contributes to guanylation and uridylation on mature miRNAs. TUT4 uridylates most miRNAs whereas TUT7 is dispensable. Removing adenylation has a marginal impact on miRNA levels. By contrast, abolishing uridylation leads to dysregulation of a set of miRNAs. Besides let-7, miR-181b and miR-222 are negatively regulated by TUT4/7 via distinct mechanisms while the miR-888 cluster is upregulated specifically by TUT7. Our results uncover the selective actions of TENTs in generating 3' isomiRs and pave the way to investigate their functions.
Asunto(s)
Proteínas de Unión al ADN , MicroARNs , Polinucleotido Adenililtransferasa , ARN Nucleotidiltransferasas , Factores de Escisión y Poliadenilación de ARNm , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , MicroARNs/genética , Polinucleotido Adenililtransferasa/genética , ARN Nucleotidiltransferasas/genética , Uridina Monofosfato/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
RNA uridylation, catalyzed by terminal uridylyl transferases (TUTases), represents a conserved and widespread posttranscriptional RNA modification in eukaryotes that affects RNA metabolism. In plants, several TUTases, including HEN1 SUPPRESSOR 1 (HESO1) and UTP: RNA URIDYLYLTRANSFERASE (URT1), have been characterized through genetic and biochemical approaches. However, little is known about their physiological significance during plant development. Here, we show that HESO1 and URT1 act cooperatively with the cytoplasmic 3'-5' exoribonucleolytic machinery component SUPERKILLER 2 (SKI2) to regulate photosynthesis through RNA surveillance of the Calvin cycle gene TRANSKETOLASE 1 (TKL1) in Arabidopsis. Simultaneous dysfunction of HESO1, URT1, and SKI2 resulted in leaf etiolation and reduced photosynthetic efficiency. In addition, we detected massive illegitimate short interfering RNAs (siRNAs) from the TKL1 locus in heso1 urt1 ski2, accompanied by reduced TKL1/2 expression and attenuated TKL activities. Consequently, the metabolic analysis revealed that the abundance of many Calvin cycle intermediates is dramatically disturbed in heso1 urt1 ski2. Importantly, all these molecular and physiological defects were largely rescued by the loss-of-function mutation in RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), demonstrating illegitimate siRNA-mediated TKL silencing. Taken together, our results suggest that HESO1- and URT1-mediated RNA uridylation connects to the cytoplasmic RNA degradation pathway for RNA surveillance, which is crucial for TKL expression and photosynthesis in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Fotosíntesis , ARN Nucleotidiltransferasas , Estabilidad del ARN , ARN Interferente Pequeño , Transcetolasa , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Nucleotidiltransferasas/metabolismo , Fotosíntesis/genética , ARN Helicasas/metabolismo , ARN Nucleotidiltransferasas/genética , ARN Nucleotidiltransferasas/metabolismo , Estabilidad del ARN/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transcetolasa/genética , Transcetolasa/metabolismo , Uridina/metabolismoRESUMEN
The germ line provides an excellent in vivo system to study the regulation and function of RNP granules. Germ granules are conserved germ line-specific RNP granules that are positioned in the Caenorhabditis elegans adult gonad to function in RNA maintenance, regulation, and surveillance. In Caenorhabditis elegans, when oogenesis undergoes extended meiotic arrest, germ granule proteins and other RNA-binding proteins assemble into much larger RNP granules whose hypothesized function is to regulate RNA metabolism and maintain oocyte quality. To gain insight into the function of oocyte RNP granules, in this report, we characterize distinct phases for four protein components of RNP granules in arrested oocytes. We find that the RNA-binding protein PGL-1 is dynamic and has liquid-like properties, while the intrinsically disordered protein MEG-3 has gel-like properties, similar to the properties of the two proteins in small germ granules of embryos. We find that MEX-3 exhibits several gel-like properties but is more dynamic than MEG-3, while CGH-1 is dynamic but does not consistently exhibit liquid-like characteristics and may be an intermediate phase within RNP granules. These distinct phases of RNA-binding proteins correspond to, and may underlie, differential responses to stress. Interestingly, in oocyte RNP granules, MEG-3 is not required for the condensation of PGL-1 or other RNA-binding proteins, which differs from the role of MEG-3 in small, embryonic germ granules. Lastly, we show that the PUF-5 translational repressor appears to promote MEX-3 and MEG-3 condensation into large RNP granules; however, this role may be associated with regulation of oogenesis.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , Oocitos/metabolismo , ARN/metabolismo , ARN Nucleotidiltransferasas/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Aims: Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent malignancies with unfavorable clinical outcomes and limited therapeutic methods. As a key enzyme in RNA metabolism, debranching RNA Lariats 1 (DBR1) is involved in intron turnover and biogenesis of noncoding RNA. Although cancer cells often show disorder of nucleic acid metabolism, it is unclear whether DBR1 has any effect on the carcinogenesis and progression of ESCC. Methods: Here we detected DBR1 expression in 112 ESCC samples by immunohistochemistry and analyzed its correlation with clinical parameters and survival. Results: DBR1 is mainly located in the nucleus of ESCC tissue. And DBR1 was associated with several malignant clinical features in patients, including tumor location (χ2 = 9.687, P = .021), pathologic T stage (χ2 = 5.771, P = .016), lymph node metastasis (χ2 = 8.215, P = .004) and N classification (χ2 = 10.066, P = .018). Moreover, Kaplan-Meier analysis showed that ESCC patients harboring lower DBR1 expression had a worse prognosis in comparison with those with higher DBR1 expression (P = .005). Univariate and multivariate Cox proportional hazards regression analyses indicated that decreased DBR1 might act as an independent predictor of poor prognosis for ESCC patients. Conclusion: Abnormal RNA metabolism might play a critical role in promoting the progression of ESCC, and DBR1 may be a promising potential biomarker for predicting the prognosis of ESCC patients.
Asunto(s)
Biomarcadores de Tumor , Carcinoma de Células Escamosas de Esófago , ARN Nucleotidiltransferasas , Biomarcadores de Tumor/genética , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/diagnóstico , Carcinoma de Células Escamosas de Esófago/genética , Humanos , Estimación de Kaplan-Meier , Pronóstico , Modelos de Riesgos Proporcionales , ARN , ARN Nucleotidiltransferasas/genética , ARN Nucleotidiltransferasas/metabolismoRESUMEN
The mechanisms of NASH development in the context of age and genetics are not fully elucidated. This study investigates the age-dependent liver defects during NASH development in mice with heterozygous deletion of Pcyt2 (Pcyt2+/-), the rate limiting enzyme in phosphatidylethanolamine (PE) synthesis. Further, the therapeutic potential of Pcyt2 substrate, phosphoethanolamine (PEtn), is examined. Pcyt2+/- were investigated at 2 and 6-8 months (mo) of age and in addition, 6-mo old Pcyt2+/- with developed NASH were supplemented with PEtn for 8 weeks and glucose and fatty acid metabolism, insulin signaling, and inflammation were examined. Heterozygous ablation of Pcyt2 causes changes in liver metabolic regulators from young age, prior to the development of liver disease which does not occur until adulthood. Only older Pcyt2+/- experiences perturbed glucose and fatty acid metabolism. Older Pcyt2+/- liver develops NASH characterized by increased glucose production, accumulation of TAG and glycogen, and increased inflammation. Supplementation with PEtn reverses Pcyt2+/- steatosis, inflammation, and other aspects of NASH, showing that was directly caused by Pcyt2 deficiency. Pcyt2 deficiency is a novel mechanism of metabolic dysregulation due to reduced membrane ethanolamine phospholipid synthesis, and the metabolite PEtn offers therapeutic potential for NASH reversion.
Asunto(s)
Etanolaminas/administración & dosificación , Resistencia a la Insulina , Enfermedad del Hígado Graso no Alcohólico/enzimología , ARN Nucleotidiltransferasas/deficiencia , Envejecimiento , Animales , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Ratones , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , ARN Nucleotidiltransferasas/genéticaRESUMEN
The human terminal uridyl transferases TUT4 and TUT7 (TUT4/7) catalyze the additions of uridines at the 3' end of RNAs, including the precursors of the tumor suppressor miRNA let-7 upon recruitment by the oncoprotein LIN28A. As a consequence, let-7 family miRNAs are down-regulated. Disruption of this TUT4/7 activity inhibits tumorigenesis. Hence, targeting TUT4/7 could be a potential anticancer therapy. In this study, we investigate TUT4/7-mediated RNA regulation in two cancer cell lines by establishing catalytic knockout models. Upon TUT4/7 mutation, we observe a significant reduction in miRNA uridylation, which results in defects in cancer cell properties such as cell proliferation and migration. With the loss of TUT4/7-mediated miRNA uridylation, the uridylated miRNA variants are replaced by adenylated isomiRs. Changes in miRNA modification profiles are accompanied by deregulation of expression levels in specific cases. Unlike let-7s, most miRNAs do not depend on LIN28A for TUT4/7-mediated regulation. Additionally, we identify TUT4/7-regulated cell-type-specific miRNA clusters and deregulation in their corresponding mRNA targets. Expression levels of miR-200c-3p and miR-141-3p are regulated by TUT4/7 in a cancer cell-type-specific manner. Subsequently, BCL2, which is a well-established target of miR-200c is up-regulated. Therefore, TUT4/7 loss causes deregulation of miRNA-mRNA networks in a cell-type-specific manner. Understanding of the underlying biology of such cell-type-specific deregulation will be an important aspect of targeting TUT4/7 for potential cancer therapies.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Neoplasias/metabolismo , ARN Nucleotidiltransferasas/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , MicroARNs/genética , Neoplasias/genética , ARN Nucleotidiltransferasas/genética , Procesamiento Postranscripcional del ARNRESUMEN
Previous studies indicated that the P-body components, CGH-1 and EDC-3 may play a crucial role in the regulation of lifespan in Caenorhabditis elegans. Homo sapiens DDX6 or Saccharomyces cerevisiae Dhh1p (CGH-1 in C. elegans) could form complexes with EDC3 (Edc3p in yeast), respectively, which is significant for translation inhibition and mRNA decay. However, it is currently unclear how CGH-1 can be recognized by EDC-3 in C. elegans. Here, we provided structural and biochemical insights into the interaction between CGH-1 and EDC-3. Combined with homology modeling, mutation, and ITC assays, we uncovered an interface between CGH-1 RecA2 domain and EDC-3 FDF-FEK. Additionally, GST-pulldown and co-localization experiments confirmed the interaction between CGH-1 and EDC-3 in vitro and in vivo. We also analyzed PATR-1-binding interface on CGH-1 RecA2 by ITC assays. Moreover, we unveiled the similarity and differences of the binding mode between EDC-3 and CAR-1 or PATR-1. Taken together, these findings provide insights into the recognition of DEAD-box protein CGH-1 by EDC-3 FDF-FEK motif, suggesting important functional implications.
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/embriología , ARN Helicasas/química , ARN Nucleotidiltransferasas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Humanos , Unión Proteica , ARN Helicasas/genética , ARN Nucleotidiltransferasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Noncanonical poly(A) polymerases PAPD5 and PAPD7 (PAPD5/7) stabilize hepatitis B virus (HBV) RNA via the interaction with the viral posttranscriptional regulatory element (PRE), representing new antiviral targets to control HBV RNA metabolism, hepatitis B surface antigen (HBsAg) production, and viral replication. Inhibitors targeting these proteins are being developed as antiviral therapies; therefore, it is important to understand how PAPD5/7 coordinate to stabilize HBV RNA. Here, we utilized a potent small-molecule AB-452 as a chemical probe, along with genetic analyses to dissect the individual roles of PAPD5/7 in HBV RNA stability. AB-452 inhibits PAPD5/7 enzymatic activities and reduces HBsAg both in vitro (50% effective concentration [EC50] ranged from 1.4 to 6.8 nM) and in vivo by 0.94 log10. Our genetic studies demonstrate that the stem-loop alpha sequence within PRE is essential for both maintaining HBV poly(A) tail integrity and determining sensitivity toward the inhibitory effect of AB-452. Although neither single knockout (KO) of PAPD5 nor PAPD7 reduces HBsAg RNA and protein production, PAPD5 KO does impair poly(A) tail integrity and confers partial resistance to AB-452. In contrast, PAPD7 KO did not result in any measurable changes within the HBV poly(A) tails, but cells with both PAPD5 and PAPD7 KO show reduced HBsAg production and conferred complete resistance to AB-452 treatment. Our results indicate that PAPD5 plays a dominant role in stabilizing viral RNA by protecting the integrity of its poly(A) tail, while PAPD7 serves as a second line of protection. These findings inform PAPD5-targeted therapeutic strategies and open avenues for further investigating PAPD5/7 in HBV replication. IMPORTANCE Chronic hepatitis B affects more than 250 million patients and is a major public health concern worldwide. HBsAg plays a central role in maintaining HBV persistence, and as such, therapies that aim at reducing HBsAg through destabilizing or degrading HBV RNA have been extensively investigated. Besides directly degrading HBV transcripts through antisense oligonucleotides or RNA silencing technologies, small-molecule compounds targeting host factors such as the noncanonical poly(A) polymerase PAPD5 and PAPD7 have been reported to interfere with HBV RNA metabolism. Herein, our antiviral and genetic studies using relevant HBV infection and replication models further characterize the interplays between the cis element within the viral sequence and the trans elements from the host factors. PAPD5/7-targeting inhibitors, with oral bioavailability, thus represent an opportunity to reduce HBsAg through destabilizing HBV RNA.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Virus de la Hepatitis B/genética , Hepatitis B/virología , ARN Nucleotidiltransferasas/metabolismo , Estabilidad del ARN , ARN Viral/química , Replicación Viral , Animales , Antivirales/farmacología , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Proteínas Cromosómicas no Histona/genética , ADN Polimerasa Dirigida por ADN/genética , Inhibidores Enzimáticos/farmacología , Células Hep G2 , Hepatitis B/genética , Hepatitis B/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Nucleotidiltransferasas/antagonistas & inhibidores , ARN Nucleotidiltransferasas/genética , ARN Viral/genéticaRESUMEN
Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3' terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development.