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1.
Nat Plants ; 7(2): 184-197, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33495557

RESUMO

DNA methylation is an important epigenetic gene regulatory mechanism conserved in eukaryotes. Emerging evidence shows DNA methylation alterations in response to environmental cues. However, the mechanism of how cells sense these signals and reprogramme the methylation landscape is poorly understood. Here, we uncovered a connection between ultraviolet B (UVB) signalling and DNA methylation involving UVB photoreceptor (UV RESISTANCE LOCUS 8 (UVR8)) and a de novo DNA methyltransferase (DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2)) in Arabidopsis. We demonstrated that UVB acts through UVR8 to inhibit DRM2-mediated DNA methylation and transcriptional de-repression. Interestingly, DNA transposons with high DNA methylation are more sensitive to UVB irradiation. Mechanistically, UVR8 interacts with and negatively regulates DRM2 by preventing its chromatin association and inhibiting the methyltransferase activity. Collectively, this study identifies UVB as a potent inhibitor of DNA methylation and provides mechanistic insights into how signalling transduction cascades intertwine with chromatin to guide genome functions.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Metilação de DNA , Regulação da Expressão Gênica de Plantas , Metiltransferases/genética , Metiltransferases/metabolismo , Raios Ultravioleta , Arabidopsis/metabolismo , Genes de Plantas , Variação Genética , Genótipo , Mutação , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
2.
New Phytol ; 229(6): 3303-3317, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33216996

RESUMO

DNA methylation plays crucial roles in cellular development and stress responses through gene regulation and genome stability control. Precise regulation of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), the de novo Arabidopsis DNA methyltransferase, is crucial to maintain DNA methylation homeostasis to ensure genome integrity. Compared with the extensive studies on DRM2 targeting mechanisms, little information is known regarding the quality control of DRM2 itself. Here, we conducted yeast two-hybrid screen assay and identified an E3 ligase, COP9 INTERACTING F-BOX KELCH 1 (CFK1), as a novel DRM2-interacting partner and targets DRM2 for degradation via the ubiquitin-26S proteasome pathway in Arabidopsis thaliana. We also performed whole genome bisulfite sequencing (BS-seq) to determine the biological significance of CFK1-mediated DRM2 degradation. Loss-of-function CFK1 leads to increased DRM2 protein abundance and overexpression of CFK1 showed reduced DRM2 protein levels. Consistently, CFK1 overexpression induces genome-wide CHH hypomethylation and transcriptional de-repression at specific DRM2 target loci. This study uncovered a distinct mechanism regulating de novo DNA methyltransferase by CFK1 to control DNA methylation level.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas F-Box , Metiltransferases , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , DNA , Metilação de DNA/genética , Proteínas F-Box/genética , Regulação da Expressão Gênica de Plantas , Metiltransferases/metabolismo
3.
Leukemia ; 33(3): 671-685, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30206308

RESUMO

The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function NOTCH1 mutations are prevalent in human T-cell leukemia, while loss of Notch signaling is reported in myeloid leukemias. Here, we report a novel oncogenic function of Notch signaling in oncogenic Kras-induced myeloproliferative neoplasm (MPN). We find that downregulation of Notch signaling in hematopoietic cells via DNMAML expression or Pofut1 deletion significantly blocks MPN development in KrasG12D mice in a cell-autonomous manner. Further mechanistic studies indicate that inhibition of Notch signaling upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in KrasG12D cells. Moreover, mitochondrial metabolism is greatly enhanced in KrasG12D cells but significantly reprogrammed by DNMAML close to that in control cells. Consequently, cell proliferation and expanded myeloid compartment in KrasG12D mice are significantly reduced. Consistent with these findings, combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target both ERK signaling and aberrant metabolism in oncogenic Ras-driven myeloid leukemia.


Assuntos
Regulação para Baixo/genética , Leucemia Mieloide/genética , Sistema de Sinalização das MAP Quinases/genética , Transtornos Mieloproliferativos/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores Notch/genética , Transdução de Sinais/genética , Animais , Proliferação de Células/genética , Citocinas/genética , Fosfatase 1 de Especificidade Dupla/genética , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Mutação/genética , Fosforilação Oxidativa , Regulação para Cima/genética
4.
Nat Genet ; 50(9): 1247-1253, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30082787

RESUMO

The ability of cells to perceive and translate versatile cues into differential chromatin and transcriptional states is critical for many biological processes1-5. In plants, timely transition to a flowering state is crucial for successful reproduction6-9. EARLY BOLTING IN SHORT DAY (EBS) is a negative transcriptional regulator that prevents premature flowering in Arabidopsis thaliana10,11. We found that EBS contains bivalent bromo-adjacent homology (BAH)-plant homeodomain (PHD) reader modules that bind H3K27me3 and H3K4me3, respectively. We observed co-enrichment of a subset of EBS-associated genes with H3K4me3, H3K27me3, and Polycomb repressor complex 2 (PRC2). Notably, EBS adopted an autoinhibition mode to mediate its switch in binding preference between H3K27me3 and H3K4me3. This binding balance was critical because disruption of either EBS-H3K27me3 or EBS-H3K4me3 interaction induced early floral transition. Our results identify a bivalent chromatin reader capable of recognizing two antagonistic histone marks, and we propose a distinct mechanism of interaction between active and repressive chromatin states.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Flores/genética , Genes Reguladores/genética , Histonas/genética , Cromatina/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Homeodomínio/genética , Transcrição Gênica/genética
5.
Nat Commun ; 9(1): 2425, 2018 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-29930355

RESUMO

The ability of a cell to dynamically switch its chromatin between different functional states constitutes a key mechanism regulating gene expression. Histone mark "readers" display distinct binding specificity to different histone modifications and play critical roles in regulating chromatin states. Here, we show a plant-specific histone reader SHORT LIFE (SHL) capable of recognizing both H3K27me3 and H3K4me3 via its bromo-adjacent homology (BAH) and plant homeodomain (PHD) domains, respectively. Detailed biochemical and structural studies suggest a binding mechanism that is mutually exclusive for either H3K4me3 or H3K27me3. Furthermore, we show a genome-wide co-localization of SHL with H3K27me3 and H3K4me3, and that BAH-H3K27me3 and PHD-H3K4me3 interactions are important for SHL-mediated floral repression. Together, our study establishes BAH-PHD cassette as a dual histone methyl-lysine binding module that is distinct from others in recognizing both active and repressive histone marks.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cromatina/metabolismo , Regulação da Expressão Gênica de Plantas , Histonas/metabolismo , Arabidopsis/metabolismo , Código das Histonas , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/fisiologia , Metilação , Modelos Genéticos
6.
Nat Commun ; 9(1): 630, 2018 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-29434220

RESUMO

The dynamic incorporation of histone variants influences chromatin structure and many biological processes. In Arabidopsis, the canonical variant H3.1 differs from H3.3 in four residues, one of which (H3.1Phe41) is unique and conserved in plants. However, its evolutionary significance remains unclear. Here, we show that Phe41 first appeared in H3.1 in ferns and became stable during land plant evolution. Unlike H3.1, which is specifically enriched in silent regions, H3.1F41Y variants gain ectopic accumulation at actively transcribed regions. Reciprocal tail and core domain swap experiments between H3.1 and H3.3 show that the H3.1 core, while necessary, is insufficient to restrict H3.1 to silent regions. We conclude that the vascular-plant-specific Phe41 is critical for H3.1 genomic distribution and may act collaboratively with the H3.1 core to regulate deposition patterns. This study reveals that Phe41 may have evolved to provide additional regulation of histone deposition in plants.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Genoma de Planta , Histonas/química , Histonas/metabolismo , Motivos de Aminoácidos , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica de Plantas , Histonas/genética
7.
Plant Cell ; 30(1): 134-152, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29343504

RESUMO

Ribosome biogenesis is a fundamental process required for all cellular activities. Histone deacetylases play critical roles in many biological processes including transcriptional repression and rDNA silencing. However, their function in pre-rRNA processing remains poorly understood. Here, we discovered a previously uncharacterized role of Arabidopsis thaliana histone deacetylase HD2C in pre-rRNA processing via both canonical and noncanonical manners. HD2C interacts with another histone deacetylase HD2B and forms homo- and/or hetero-oligomers in the nucleolus. Depletion of HD2C and HD2B induces a ribosome-biogenesis deficient phenotype and aberrant accumulation of 18S pre-rRNA intermediates. Our genome-wide analysis revealed that HD2C binds and represses the expression of key genes involved in ribosome biogenesis. Using RNA immunoprecipitation and sequencing, we further uncovered a noncanonical mechanism of HD2C directly associating with pre-rRNA and small nucleolar RNAs to regulate rRNA methylation. Together, this study reveals a multifaceted role of HD2C in ribosome biogenesis and provides mechanistic insights into how histone deacetylases modulate rRNA maturation at the transcriptional and posttranscriptional levels.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Histona Desacetilases/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico/genética , Acetilação , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sequência de Bases , Deleção de Genes , Genes de Plantas , Pleiotropia Genética , Histona Desacetilases/química , Histona Desacetilases/genética , Histonas/metabolismo , Lisina/metabolismo , Metilação , Modelos Biológicos , Biogênese de Organelas , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Ribossomos/metabolismo , Nicotiana/genética
8.
Plant Physiol ; 173(4): 2243-2252, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28202597

RESUMO

Epigenetic modifications play critical roles in diverse biological processes. Histone Lys-to-Met (K-to-M) mutations act as gain-of-function mutations to inhibit a wide range of histone methyltransferases and are thought to promote tumorigenesis. However, it is largely unknown whether K-to-M mutations impact organismal development. Using Arabidopsis (Arabidopsis thaliana) as a model system, we discovered that a transgene exogenously expressing histone 3 Lys-36 to Met mutation (K36M) acts in a dominant-negative manner to cause global reduction of H3K36 methylation. Remarkably, this dominant repressive activity is dosage-dependent and causes strong developmental perturbations including extreme branching and early flowering by affecting the expression of genes involved in developmental and metabolic processes. Besides the established pathological roles of K-to-M mutations in tumor cells, we demonstrate a physiological outcome for K-to-M induced H3K36 hypomethylation. This study provides evidence for a conserved dominant-negative inhibitory role of histone K-to-M mutation across the plant and animal kingdoms. We also highlight the unique ability of K36M mutations to alter plant developmental processes leading to severe pleiotropic phenotypes. Finally, our data suggests K-to-M mutations may provide a useful strategy for altering epigenetic landscapes in organisms where histone methyltransferases are uncharacterized.


Assuntos
Proteínas de Arabidopsis/genética , Histonas/genética , Lisina/genética , Metionina/genética , Mutação , Substituição de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Epigênese Genética , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Pleiotropia Genética , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Immunoblotting , Lisina/metabolismo , Metionina/metabolismo , Metilação , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
9.
Elife ; 52016 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-27873573

RESUMO

Leaf senescence is an essential part of the plant lifecycle during which nutrients are re-allocated to other tissues. The regulation of leaf senescence is a complex process. However, the underlying mechanism is poorly understood. Here, we uncovered a novel and the pivotal role of Arabidopsis HDA9 (a RPD3-like histone deacetylase) in promoting the onset of leaf senescence. We found that HDA9 acts in complex with a SANT domain-containing protein POWERDRESS (PWR) and transcription factor WRKY53. Our genome-wide profiling of HDA9 occupancy reveals that HDA9 directly binds to the promoters of key negative regulators of senescence and this association requires PWR. Furthermore, we found that PWR is important for HDA9 nuclear accumulation. This study reveals an uncharacterized epigenetic complex involved in leaf senescence and provides mechanistic insights into how a histone deacetylase along with a chromatin-binding protein contribute to a robust regulatory network to modulate the onset of plant aging.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Histona Desacetilases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/metabolismo , Multimerização Proteica
10.
Epigenetics ; 10(11): 1044-53, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26646900

RESUMO

Histone acetylation and deacetylation are key epigenetic gene regulatory mechanisms that play critical roles in eukaryotes. Acetylation of histone 4 lysine 16 (H4K16ac) is implicated in many cellular processes. However, its biological function and relationship with transcription are largely unexplored in plants. We generated first genome-wide high-resolution maps of H4K16ac in Arabidopsis thaliana and Oryza sativa. We showed that H4K16ac is preferentially enriched around the transcription start sites and positively correlates with gene expression levels. Co-existence of H4K16ac and H3K23ac is correlated with high gene expression levels, suggesting a potentially combinatorial effect of H4K16ac and H3K23ac histone 3 lysine 23 acetylation on gene expression. Our data further revealed that while genes enriched with both H4K16ac and H3K23ac are ubiquitously expressed, genes enriched with only H4K16ac or H3K23ac showed significantly distinct expression patterns in association with particular developmental stages. Unexpectedly, and unlike in Arabidopsis, there are significant levels of both H4K16ac and H3K23ac in the lowly expressed genes in rice. Furthermore, we found that H4K16ac-enriched genes are associated with different biological processes in Arabidopsis and rice, suggesting a potentially species-specific role of H4K16ac in plants. Together, our genome-wide profiling reveals the conserved and unique distribution patterns of H4K16ac and H3K23ac in Arabidopsis and rice and provides a foundation for further understanding their function in plants.


Assuntos
Arabidopsis/genética , Mapeamento Cromossômico/métodos , Histonas/metabolismo , Oryza/genética , Acetilação , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Transcrição Gênica
11.
J Virol ; 86(18): 10059-69, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22787202

RESUMO

Hepadnaviral covalently closed circular DNA (cccDNA) exists as an episomal minichromosome in the nucleus of virus-infected hepatocytes, and serves as the transcriptional template for the synthesis of viral mRNAs. To obtain insight on the structure of hepadnaviral cccDNA minichromosomes, we utilized ducks infected with the duck hepatitis B virus (DHBV) as a model and determined the in vivo nucleosome distribution pattern on viral cccDNA by the micrococcal nuclease (MNase) mapping and genome-wide PCR amplification of isolated mononucleosomal DHBV DNA. Several nucleosome-protected sites in a region of the DHBV genome [nucleotides (nt) 2000 to 2700], known to harbor various cis transcription regulatory elements, were consistently identified in all DHBV-positive liver samples. In addition, we observed other nucleosome protection sites in DHBV minichromosomes that may vary among individual ducks, but the pattern of MNase mapping in those regions is transmittable from the adult ducks to the newly infected ducklings. These results imply that the nucleosomes along viral cccDNA in the minichromosomes are not random but sequence-specifically positioned. Furthermore, we showed in ducklings that a significant portion of cccDNA possesses a few negative superhelical turns, suggesting the presence of intermediates of viral minichromosomes assembled in the liver, where dynamic hepatocyte growth and cccDNA formation occur. This study supplies the initial framework for the understanding of the overall complete structure of hepadnaviral cccDNA minichromosomes.


Assuntos
DNA Circular/genética , DNA Viral/genética , Vírus da Hepatite B do Pato/genética , Nucleossomos/virologia , Animais , Sequência de Bases , Sítios de Ligação/genética , Mapeamento Cromossômico , DNA Circular/química , DNA Circular/metabolismo , DNA Viral/química , DNA Viral/metabolismo , Patos , Genoma Viral , Infecções por Hepadnaviridae/virologia , Vírus da Hepatite B do Pato/patogenicidade , Vírus da Hepatite B do Pato/fisiologia , Hepatite Viral Animal/virologia , Fígado/virologia , Nuclease do Micrococo , Plasmídeos/genética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo
12.
Retrovirology ; 8: 36, 2011 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-21569500

RESUMO

BACKGROUND: MicroRNA (miRNA)-mediated RNA silencing is integral to virtually every cellular process including cell cycle progression and response to virus infection. The interplay between RNA silencing and HIV-1 is multifaceted, and accumulating evidence posits a strike-counterstrike interface that alters the cellular environment to favor virus replication. For instance, miRNA-mediated RNA silencing of HIV-1 translation is antagonized by HIV-1 Tat RNA silencing suppressor activity. The activity of HIV-1 accessory proteins Vpr/Vif delays cell cycle progression, which is a process prominently modulated by miRNA. The expression profile of cellular miRNA is altered by HIV-1 infection in both cultured cells and clinical samples. The open question stands of what, if any, is the contribution of Tat RNA silencing suppressor activity or Vpr/Vif activity to the perturbation of cellular miRNA by HIV-1. RESULTS: Herein, we compared the perturbation of miRNA expression profiles of lymphocytes infected with HIV-1(NL4-3) or derivative strains that are deficient in Tat RNA silencing suppressor activity (Tat K51A substitution) or ablated of the vpr/vif open reading frames. Microarrays recapitulated the perturbation of the cellular miRNA profile by HIV-1 infection. The miRNA expression trends overlapped ~50% with published microarray results on clinical samples from HIV-1 infected patients. Moreover, the number of miRNA perturbed by HIV-1 was largely similar despite ablation of Tat RSS activity and Vpr/Vif; however, the Tat RSS mutation lessened HIV-1 downregulation of twenty-two miRNAs. CONCLUSIONS: Our study identified miRNA expression changes attributable to Tat RSS activity in HIV-1(NL4-3). The results accomplish a necessary step in the process to understand the interface of HIV-1 with host RNA silencing activity. The overlap in miRNA expression trends observed between HIV-1 infected CEMx174 lymphocytes and primary cells supports the utility of cultured lymphocytes as a tractable model to investigate interplay between HIV-1 and host RNA silencing. The subset of miRNA determined to be perturbed by Tat RSS in HIV-1 infection provides a focal point to define the gene networks that shape the cellular environment for HIV-1 replication.


Assuntos
HIV-1/imunologia , HIV-1/patogenicidade , Linfócitos/imunologia , Linfócitos/virologia , MicroRNAs/biossíntese , Fatores de Virulência/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Células Cultivadas , Deleção de Genes , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Análise em Microsséries , Fatores de Virulência/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo
13.
Proc Natl Acad Sci U S A ; 106(2): 605-10, 2009 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-19122141

RESUMO

The RNA silencing pathway is an intracellular innate response to virus infections and retro-transposons. Many plant viruses counter this host restriction by RNA silencing suppressor (RSS) activity of a double-stranded RNA-binding protein, e.g., tomato bushy stunt virus P19. Here, we demonstrate P19 and HIV-1 Tat function across the plant and animal kingdoms and suppress a common step in RNA silencing that is downstream of small RNA maturation. Our experiments reveal that RNA silencing in HIV-1 infected human cells severely attenuates the translational output of the unspliced HIV-1 gag mRNA, and possibly all HIV-1 transcripts. The attenuation in gag mRNA translation is exacerbated by K51A substitution in the Tat double-stranded RNA-binding domain. Tat, plant virus RSS, or Dicer downregulation rescues robust gag translation and bolsters HIV-1 virion production. The reversal of HIV-1 translation repression by plant RSS supports the recent finding in Arabidopsis that plant miRNAs operate by translational inhibition. Our results identify common features between RNA silencing suppression of plant and animal viruses. We suggest that RNA silencing-mediated translation repression plays a strategic role in determining the viral set-point in a newly HIV-1-infected patient.


Assuntos
HIV-1/patogenicidade , Imunidade Inata , Interferência de RNA/imunologia , Replicação Viral , Produtos do Gene tat do Vírus da Imunodeficiência Humana/fisiologia , Linhagem Celular , Produtos do Gene gag/biossíntese , Infecções por HIV , HIV-1/genética , Humanos , Vírus de Plantas/genética , Vírus de Plantas/patogenicidade , Biossíntese de Proteínas , RNA Viral , Proteínas Virais/fisiologia
14.
J Virol ; 80(17): 8566-81, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16912306

RESUMO

RNA-templated RNA replication is essential for viral or viroid infection, as well as for regulation of cellular gene expression. Specific RNA motifs likely regulate various aspects of this replication. Viroids of the Pospiviroidae family, as represented by the Potato spindle tuber viroid (PSTVd), replicate in the nucleus by utilizing DNA-dependent RNA polymerase II. We investigated the role of the loop E (sarcin/ricin) motif of the PSTVd genomic RNA in replication. A tertiary-structural model of this motif, inferred by comparative sequence analysis and comparison with nuclear magnetic resonance and X-ray crystal structures of loop E motifs in other RNAs, is presented in which core non-Watson-Crick base pairs are precisely specified. Isostericity matrix analysis of these base pairs showed that the model accounts for the reported natural sequence variations and viable experimental mutations in loop E motifs of PSTVd and other viroids. Furthermore, isostericity matrix analysis allowed us to design disruptive, as well as compensatory, mutations of PSTVd loop E. Functional analyses of such mutants by in vitro and in vivo experiments demonstrated that loop E structural integrity is crucial for replication, specifically during transcription. Our results suggest that the PSTVd loop E motif exists and functions in vivo and provide loss-of-function genetic evidence for the essential role of a viroid RNA three-dimensional motif in rolling-circle replication. The use of isostericity matrix analysis of non-Watson-Crick base pairing to rationalize mutagenesis of tertiary motifs and systematic in vitro and in vivo functional assays of mutants offers a novel, comprehensive approach to elucidate the tertiary-structure-function relationships for RNA motifs of general biological significance.


Assuntos
RNA Viral/química , RNA Viral/metabolismo , Solanum tuberosum/virologia , Viroides/fisiologia , Replicação Viral , Pareamento de Bases , Sequência de Bases , Dados de Sequência Molecular , Mutagênese , Conformação de Ácido Nucleico , Vírus de Plantas/química , Vírus de Plantas/genética , Vírus de Plantas/metabolismo , Protoplastos/virologia , RNA Viral/genética , Nicotiana/virologia , Transcrição Gênica , Viroides/química , Viroides/metabolismo , Viroides/patogenicidade
15.
Nat Struct Mol Biol ; 13(6): 509-16, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16680162

RESUMO

RNA helicase A (RHA) is a highly conserved DEAD-box protein that activates transcription, modulates RNA splicing and binds the nuclear pore complex. The life cycle of typical mRNA involves RNA processing and translation after ribosome scanning of a relatively unstructured 5' untranslated region (UTR). The precursor RNAs of retroviruses and selected cellular genes harbor a complex 5' UTR and use a yet-to-be-identified host post-transcriptional effector to stimulate efficient translation. Here we show that RHA recognizes a structured 5'-terminal post-transcriptional control element (PCE) of a retrovirus and the JUND growth-control gene. RHA interacts with PCE RNA in the nucleus and cytoplasm, facilitates polyribosome association and is necessary for its efficient translation. Our results reveal a previously unidentified role for RHA in translation and implicate RHA as an integrative effector in the continuum of gene expression from transcription to translation.


Assuntos
Autoantígenos/metabolismo , Biossíntese de Proteínas , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Regiões 5' não Traduzidas , Sequência de Bases , RNA Helicases DEAD-box , Regulação para Baixo , Dados de Sequência Molecular , Proteínas de Neoplasias , Proteínas Repressoras/genética
16.
Yi Chuan ; 24(5): 555-8, 2002 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-16135449

RESUMO

Using the technique of natural aging,chloroform and EDTA treatment,well-resolved G-banding patterns were successfully obtained in Rana plancyt's mitosis chromosome firstly. The G-banding patterns appeared distinctive. When analyzing some homologous chromosomes from the same metaphase figures and four macrochromosomes from different metaphase figures,the characteristic and the number of the bands were well matched. The method in this study is also economic and simple and the result is stable and reliable. The possible mechanism of G-banding was primarily discussed. The application of this method in the cytogenetics research of Rana plancyt and other amphibians is expected.

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