RESUMO
LINE-1 retrotransposition is tightly restricted by layers of regulatory control, with epigenetic pathways being the best characterized. Looking at post-transcriptional regulation, we now show that LINE-1 mRNA 3' ends are pervasively uridylated in various human cellular models and in mouse testes. TUT4 and TUT7 uridyltransferases catalyze the modification and function in cooperation with the helicase/RNPase MOV10 to counteract the RNA chaperone activity of the L1-ORF1p retrotransposon protein. Uridylation potently restricts LINE-1 retrotransposition by a multilayer mechanism depending on differential subcellular localization of the uridyltransferases. We propose that uridine residues added by TUT7 in the cytoplasm inhibit initiation of reverse transcription of LINE-1 mRNAs once they are reimported to the nucleus, whereas uridylation by TUT4, which is enriched in cytoplasmic foci, destabilizes mRNAs. These results provide a model for the post-transcriptional restriction of LINE-1, revealing a key physiological role for TUT4/7-mediated uridylation in maintaining genome stability.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , RNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Uridina/metabolismo , Animais , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Camundongos , Proteínas Nucleares/genética , Ligação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , RNA Helicases/metabolismo , Interferência de RNA , RNA Nucleotidiltransferases/antagonistas & inibidores , RNA Nucleotidiltransferases/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Retroelementos/genéticaRESUMO
Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.
Assuntos
Encefalopatias Metabólicas Congênitas/genética , Tronco Encefálico/metabolismo , Tronco Encefálico/virologia , RNA/química , RNA/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Encefalopatias Metabólicas Congênitas/patologia , Tronco Encefálico/patologia , Encefalite Viral/genética , Escherichia coli/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/virologia , Herpesvirus Humano 1 , Humanos , Interferons/metabolismo , Íntrons/genética , Masculino , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , Fases de Leitura Aberta/genética , Linhagem , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/deficiência , RNA Nucleotidiltransferases/genética , Receptor 3 Toll-Like/metabolismo , Replicação ViralRESUMO
The modular Integrator complex is a transcription regulator that is essential for embryonic development. It attenuates coding gene expression via premature transcription termination and performs 3'-processing of non-coding RNAs. For both activities, Integrator requires endonuclease activity that is harbored by an RNA cleavage module consisting of INTS4-9-11. How correct assembly of Integrator modules is achieved remains unknown. Here, we show that BRAT1 and WDR73 are critical biogenesis factors for the human cleavage module. They maintain INTS9-11 inactive during maturation by physically blocking the endonuclease active site and prevent premature INTS4 association. Furthermore, BRAT1 facilitates import of INTS9-11 into the nucleus, where it is joined by INTS4. Final BRAT1 release requires locking of the mature cleavage module conformation by inositol hexaphosphate (IP6). Our data explain several neurodevelopmental disorders caused by BRAT1, WDR73, and INTS11 mutations as Integrator assembly defects and reveal that IP6 is an essential co-factor for cleavage module maturation.
Assuntos
Clivagem do RNA , Humanos , Células HEK293 , Ácido Fítico/metabolismo , Mutação , Núcleo Celular/metabolismo , Núcleo Celular/genética , Domínio Catalítico , Ligação Proteica , RNA NucleotidiltransferasesRESUMO
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.
Assuntos
Síndromes de Tricotiodistrofia , Animais , Camundongos , Íntrons/genética , Síndromes de Tricotiodistrofia/genética , RNA Nucleotidiltransferases/genética , Splicing de RNARESUMO
Transcription in eukaryotes produces a number of long noncoding RNAs (lncRNAs). Two of these, MALAT1 and Menß, generate a tRNA-like small RNA in addition to the mature lncRNA. The stability of these tRNA-like small RNAs and bona fide tRNAs is monitored by the CCA-adding enzyme. Whereas CCA is added to stable tRNAs and tRNA-like transcripts, a second CCA repeat is added to certain unstable transcripts to initiate their degradation. Here, we characterize how these two scenarios are distinguished. Following the first CCA addition cycle, nucleotide binding to the active site triggers a clockwise screw motion, producing torque on the RNA. This ejects stable RNAs, whereas unstable RNAs are refolded while bound to the enzyme and subjected to a second CCA catalytic cycle. Intriguingly, with the CCA-adding enzyme acting as a molecular vise, the RNAs proofread themselves through differential responses to its interrogation between stable and unstable substrates.
Assuntos
Archaeoglobus fulgidus/enzimologia , Mitocôndrias/enzimologia , RNA Nucleotidiltransferases/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Archaeoglobus fulgidus/metabolismo , Sequência de Bases , Domínio Catalítico , Humanos , Mitocôndrias/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/genética , Estabilidade de RNA , Pequeno RNA não Traduzido/metabolismoRESUMO
Uridylation occurs pervasively on mRNAs, yet its mechanism and significance remain unknown. By applying TAIL-seq, we identify TUT4 and TUT7 (TUT4/7), also known as ZCCHC11 and ZCCHC6, respectively, as mRNA uridylation enzymes. Uridylation readily occurs on deadenylated mRNAs in cells. Consistently, purified TUT4/7 selectively recognize and uridylate RNAs with short A-tails (less than â¼ 25 nt) in vitro. PABPC1 antagonizes uridylation of polyadenylated mRNAs, contributing to the specificity for short A-tails. In cells depleted of TUT4/7, the vast majority of mRNAs lose the oligo-U-tails, and their half-lives are extended. Suppression of mRNA decay factors leads to the accumulation of oligo-uridylated mRNAs. In line with this, microRNA induces uridylation of its targets, and TUT4/7 are required for enhanced decay of microRNA targets. Our study explains the mechanism underlying selective uridylation of deadenylated mRNAs and demonstrates a fundamental role of oligo-U-tail as a molecular mark for global mRNA decay.
Assuntos
Proteínas de Ligação a DNA/metabolismo , RNA Nucleotidiltransferases/metabolismo , Estabilidade de RNA , Células HeLa , Humanos , MicroRNAs/metabolismo , Poli A/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , RNA Mensageiro/metabolismo , Uridina Monofosfato/metabolismoRESUMO
Strand selection is a critical step in microRNA (miRNA) biogenesis. Although the dominant strand may change depending on cellular contexts, the molecular mechanism and physiological significance of such alternative strand selection (or "arm switching") remain elusive. Here we find miR-324 to be one of the strongly regulated miRNAs by arm switching and identify the terminal uridylyl transferases TUT4 and TUT7 to be the key regulators. Uridylation of pre-miR-324 by TUT4/7 re-positions DICER on the pre-miRNA and shifts the cleavage site. This alternative processing produces a duplex with a different terminus from which the 3' strand (3p) is selected instead of the 5' strand (5p). In glioblastoma, the TUT4/7 and 3p levels are upregulated, whereas the 5p level is reduced. Manipulation of the strand ratio is sufficient to impair glioblastoma cell proliferation. This study uncovers a role of uridylation as a molecular switch in alternative strand selection and implicates its therapeutic potential.
Assuntos
MicroRNAs/metabolismo , UDPglucose-Hexose-1-Fosfato Uridiltransferase/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , RNA Helicases DEAD-box/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Camundongos , MicroRNAs/genética , Cultura Primária de Células , RNA Nucleotidiltransferases/metabolismo , Ribonuclease III/metabolismoRESUMO
RNase III Drosha initiates microRNA (miRNA) maturation by cleaving a primary miRNA transcript and releasing a pre-miRNA with a 2 nt 3' overhang. Dicer recognizes the 2 nt 3' overhang structure to selectively process pre-miRNAs. Here, we find that, unlike prototypic pre-miRNAs (group I), group II pre-miRNAs acquire a shorter (1 nt) 3' overhang from Drosha processing and therefore require a 3'-end mono-uridylation for Dicer processing. The majority of let-7 and miR-105 belong to group II. We identify TUT7/ZCCHC6, TUT4/ZCCHC11, and TUT2/PAPD4/GLD2 as the terminal uridylyl transferases responsible for pre-miRNA mono-uridylation. The TUTs act specifically on dsRNAs with a 1 nt 3' overhang, thereby creating a 2 nt 3' overhang. Depletion of TUTs reduces let-7 levels and disrupts let-7 function. Although the let-7 suppressor, Lin28, induces inhibitory oligo-uridylation in embryonic stem cells, mono-uridylation occurs in somatic cells lacking Lin28 to promote let-7 biogenesis. Our study reveals functional duality of uridylation and introduces TUT7/4/2 as components of the miRNA biogenesis pathway.
Assuntos
Proteínas de Ligação a DNA/metabolismo , MicroRNAs/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , RNA Nucleotidiltransferases/metabolismo , Processamento Pós-Transcricional do RNA , Uridina Monofosfato/metabolismo , Sequência de Bases , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas de Ligação a RNA/metabolismo , Fatores de Poliadenilação e Clivagem de mRNARESUMO
T follicular helper (TFH) cells are crucial for B cell-mediated humoral immunity1. Although transcription factors such as BCL6 drive the differentiation of TFH cells2,3, it is unclear whether and how post-transcriptional and metabolic programs enforce TFH cell programming. Here we show that the cytidine diphosphate (CDP)-ethanolamine pathway co-ordinates the expression and localization of CXCR5 with the responses of TFH cells and humoral immunity. Using in vivo CRISPR-Cas9 screening and functional validation in mice, we identify ETNK1, PCYT2, and SELENOI-enzymes in the CDP-ethanolamine pathway for de novo synthesis of phosphatidylethanolamine (PE)-as selective post-transcriptional regulators of TFH cell differentiation that act by promoting the surface expression and functional effects of CXCR5. TFH cells exhibit unique lipid metabolic programs and PE is distributed to the outer layer of the plasma membrane, where it colocalizes with CXCR5. De novo synthesis of PE through the CDP-ethanolamine pathway co-ordinates these events to prevent the internalization and degradation of CXCR5. Genetic deletion of Pcyt2, but not of Pcyt1a (which mediates the CDP-choline pathway), in activated T cells impairs the differentiation of TFH cells, and this is associated with reduced humoral immune responses. Surface levels of PE and CXCR5 expression on B cells also depend on Pcyt2. Our results reveal that phospholipid metabolism orchestrates post-transcriptional mechanisms for TFH cell differentiation and humoral immunity, highlighting the metabolic control of context-dependent immune signalling and effector programs.
Assuntos
Imunidade Humoral , Fosfatidiletanolaminas/metabolismo , Receptores CXCR5/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Linfócitos B/imunologia , Sistemas CRISPR-Cas , Diferenciação Celular , Cistina Difosfato , Feminino , Regulação da Expressão Gênica , Humanos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfotransferases (Aceptor do Grupo Álcool) , RNA Nucleotidiltransferases , Transdução de SinaisRESUMO
Mutations in RNA-processing enzymes are increasingly linked to human disease. Telomerase RNA and related noncoding RNAs require 3' end-processing steps, including oligoadenylation. Germline mutations in poly(A)ribonuclease (PARN) cause accumulation of extended human telomerase RNA (hTR) species and precipitate dyskeratosis congenita and pulmonary fibrosis. Here, we develop nascent RNAend-seq to measure processing rates of RNA precursors. We find that mature hTR derives from extended precursors but that in PARN-mutant cells hTR maturation kinetically stalls and unprocessed precursors are degraded. Loss of poly(A)polymerase PAPD5 in PARN-mutant cells accelerates hTR maturation and restores hTR processing, indicating that oligoadenylation and deadenylation set rates of hTR maturation. The H/ACA domain mediates hTR maturation by precisely defining the 3' end, recruiting poly(A)polymerase activity, and conferring sensitivity to PARN regulation. These data reveal a feedforward circuit in which post-transcriptional oligoadenylation controls RNA maturation kinetics. Similar alterations in RNA processing rates may contribute to mechanisms of RNA-based human disease.
Assuntos
Disceratose Congênita/genética , Exorribonucleases/genética , RNA Nucleotidiltransferases/genética , RNA/genética , Telomerase/genética , Disceratose Congênita/patologia , Mutação em Linhagem Germinativa/genética , Células HeLa , Humanos , Cinética , Processamento Pós-Transcricional do RNA/genéticaRESUMO
Many microRNAs (miRNAs) exist alongside abundant miRNA isoforms (isomiRs), most of which arise from post-maturation sequence modifications such as 3' uridylation. However, the ways in which these sequence modifications affect miRNA function remain poorly understood. Here, using human miR-27a in cell lines as a model, we discovered that a nonfunctional target site unable to base-pair extensively with the miRNA seed sequence can regain function when an upstream adenosine is able to base-pair with a post-transcriptionally added uridine in the miR-27a tail. This tail-U-mediated repression (TUMR) is abolished in cells lacking the uridylation enzymes TUT4 and TUT7, indicating that uridylation alters miRNA function by modulating target recognition. We identified a set of non-canonical targets in human cells that are specifically regulated by uridylated miR-27a. We provide evidence that TUMR expands the targets of other endogenous miRNAs. Our study reveals a function of uridylated isomiRs in regulating non-canonical miRNA targets.
Assuntos
Proteínas de Ligação a DNA/genética , MicroRNAs/genética , RNA Nucleotidiltransferases/genética , Uridina/genética , Adenosina/genética , Pareamento de Bases/genética , Células HeLa , Humanos , Estabilidade de RNA , Uridina/metabolismoRESUMO
PARN loss-of-function mutations cause a severe form of the hereditary disease dyskeratosis congenita (DC). PARN deficiency affects the stability of non-coding RNAs such as human telomerase RNA (hTR), but these effects do not explain the severe disease in patients. We demonstrate that PARN deficiency affects the levels of numerous miRNAs in human cells. PARN regulates miRNA levels by stabilizing either mature or precursor miRNAs by removing oligo(A) tails added by the poly(A) polymerase PAPD5, which if remaining recruit the exonuclease DIS3L or DIS3L2 to degrade the miRNA. PARN knockdown destabilizes multiple miRNAs that repress p53 translation, which leads to an increase in p53 accumulation in a Dicer-dependent manner, thus explaining why PARN-defective patients show p53 accumulation. This work also reveals that DIS3L and DIS3L2 are critical 3' to 5' exonucleases that regulate miRNA stability, with the addition and removal of 3' end extensions controlling miRNA levels in the cell.
Assuntos
Exorribonucleases/metabolismo , MicroRNAs/metabolismo , Estabilidade de RNA , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/enzimologia , Regiões 3' não Traduzidas , Antineoplásicos/farmacologia , Sobrevivência Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Doxorrubicina/farmacologia , Etoposídeo/farmacologia , Exorribonucleases/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HeLa , Humanos , MicroRNAs/genética , Poliadenilação , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
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.
Assuntos
Citoplasma , Homeostase , RNA Mensageiro , Grânulos de Estresse , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Grânulos de Estresse/metabolismo , Citoplasma/metabolismo , Capuzes de RNA/metabolismo , Arsenitos/farmacologia , Estresse Oxidativo , Transporte Ativo do Núcleo Celular , RNA Nucleotidiltransferases/metabolismo , RNA Nucleotidiltransferases/genética , Compostos de Sódio/farmacologia , Proteína Exportina 1 , Carioferinas/metabolismo , Carioferinas/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Grânulos Citoplasmáticos/metabolismo , Estabilidade de RNA , Núcleo Celular/metabolismo , Linhagem Celular Tumoral , NucleotidiltransferasesRESUMO
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.
Assuntos
Mycobacterium tuberculosis , RNA de Transferência , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/enzimologia , Especificidade por Substrato , RNA de Transferência/metabolismo , RNA de Transferência/química , Escherichia coli/genética , Escherichia coli/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Modelos Moleculares , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Citidina/química , Citidina/metabolismo , Sítios de Ligação , Cristalografia por Raios X , RNA Nucleotidiltransferases/metabolismo , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/genéticaRESUMO
Assembly of fission yeast pericentromeric heterochromatin and generation of small interfering RNAs (siRNAs) from noncoding centromeric transcripts are mutually dependent processes. How this interdependent positive feedback loop is first triggered is a fundamental unanswered question. Here, we show that two distinct Argonaute (Ago1)-dependent pathways mediate small RNA generation. RNA-dependent RNA polymerase complex (RDRC) and Dicer act on specific noncoding RNAs to generate siRNAs by a mechanism that requires the slicer activity of Ago1 but is independent of pre-existing heterochromatin. In the absence of RDRC or Dicer, a distinct class of small RNAs, called primal small RNAs (priRNAs), associates with Ago1. priRNAs are degradation products of abundant transcripts, which bind to Ago1 and target antisense transcripts that result from bidirectional transcription of DNA repeats. Our results suggest that a transcriptome surveillance mechanism based on random association of RNA degradation products with Argonaute triggers siRNA amplification and heterochromatin assembly within DNA repeats.
Assuntos
Heterocromatina/metabolismo , Interferência de RNA , Schizosaccharomyces/metabolismo , Proteínas Argonautas , Centrômero/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Genéticos , RNA/metabolismo , Processamento de Terminações 3' de RNA , RNA Nucleotidiltransferases/metabolismo , Estabilidade de RNA , RNA Antissenso/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcrição GênicaRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Fotossíntese , RNA Nucleotidiltransferases , Estabilidade de RNA , RNA Interferente Pequeno , Transcetolase , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Nucleotidiltransferases/metabolismo , Fotossíntese/genética , RNA Helicases/metabolismo , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , Estabilidade de RNA/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transcetolase/genética , Transcetolase/metabolismo , Uridina/metabolismoRESUMO
Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5' untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3' poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3' poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1-/- mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.
Assuntos
Proteínas Cromossômicas não Histona , DNA Polimerase Dirigida por DNA , Vírus da Hepatite A , Hepatite A , Proteínas Intrinsicamente Desordenadas , RNA Nucleotidiltransferases , RNA Viral , Replicação Viral , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Proteínas Cromossômicas não Histona/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Hepatite A/tratamento farmacológico , Hepatite A/metabolismo , Hepatite A/virologia , Vírus da Hepatite A/efeitos dos fármacos , Vírus da Hepatite A/genética , Vírus da Hepatite A/fisiologia , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Camundongos , Camundongos Mutantes , RNA Nucleotidiltransferases/metabolismo , RNA Viral/biossíntese , RNA Viral/genética , Receptor de Interferon alfa e beta/genética , Replicação Viral/efeitos dos fármacosRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , RNA Nucleotidiltransferases , Humanos , Íntrons , RNA Nucleotidiltransferases/genética , RNA Nucleotidiltransferases/metabolismo , Splicing de RNA , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Ativação Enzimática/genética , Domínios Proteicos , Ligação Proteica , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Entamoeba histolytica/enzimologia , Entamoeba histolytica/genética , Metais Pesados/metabolismoRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Neoplasias/metabolismo , RNA Nucleotidiltransferases/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , MicroRNAs/genética , Neoplasias/genética , RNA Nucleotidiltransferases/genética , Processamento Pós-Transcricional do RNARESUMO
In eukaryotic cells, intron lariats produced by the spliceosome contain a 2'5' phosphodiester linkage. The RNA lariat debranching enzyme, Dbr1, is the only enzyme known to hydrolyze this bond. Dbr1 is a member of the metallophosphoesterase (MPE) family of enzymes, and recent X-ray crystal structures and biochemistry data demonstrate that Dbr1 from Entamoeba histolytica uses combinations of Mn2+, Zn2+, and Fe2+ as enzymatic cofactors. Here, we examine the kinetic properties and metal dependence of the Dbr1 homolog from Saccharomyces cerevisiae (yDbr1). Elemental analysis measured stoichiometric quantities of Fe and Zn in yDbr1 purified following heterologous expression E. coli We analyzed the ability of Fe2+, Zn2+, and Mn2+ to reconstitute activity in metal-free apoenzyme. Purified yDbr1 was highly active, turning over substrate at 5.6 sec-1, and apo-yDbr1 reconstituted with Fe2+ was the most active species, turning over at 9.2 sec-1 We treated human lymphoblastoid cells with the iron-chelator deferoxamine and measured a twofold increase in cellular lariats. These data suggest that Fe is an important biological cofactor for Dbr1 enzymes.