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1.
Hum Mol Genet ; 26(20): 3960-3972, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-29016854

RESUMO

Hypomorphic mutations in the DNA repair enzyme RNase H2 cause the neuroinflammatory autoimmune disorder Aicardi-Goutières syndrome (AGS). Endogenous nucleic acids are believed to accumulate in patient cells and instigate pathogenic type I interferon expression. However, the underlying nucleic acid species amassing in the absence of RNase H2 has not been established yet. Here, we report that murine RNase H2 knockout cells accumulated cytosolic DNA aggregates virtually indistinguishable from micronuclei. RNase H2-dependent micronuclei were surrounded by nuclear lamina and most of them contained damaged DNA. Importantly, they induced expression of interferon-stimulated genes (ISGs) and co-localized with the nucleic acid sensor cGAS. Moreover, micronuclei associated with RNase H2 deficiency were cleared by autophagy. Consequently, induction of autophagy by pharmacological mTOR inhibition resulted in a significant reduction of cytosolic DNA and the accompanied interferon signature. Autophagy induction might therefore represent a viable therapeutic option for RNase H2-dependent disease. Endogenous retroelements have previously been proposed as a source of self-nucleic acids triggering inappropriate activation of the immune system in AGS. We used human RNase H2-knockout cells generated by CRISPR/Cas9 to investigate the impact of RNase H2 on retroelement propagation. Surprisingly, replication of LINE-1 and Alu elements was blunted in cells lacking RNase H2, establishing RNase H2 as essential host factor for the mobilisation of endogenous retrotransposons.


Assuntos
Doenças Autoimunes do Sistema Nervoso/enzimologia , Micronúcleo Germinativo/enzimologia , Malformações do Sistema Nervoso/enzimologia , Ribonuclease H/deficiência , Animais , Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/metabolismo , Doenças Autoimunes do Sistema Nervoso/patologia , Autofagia/genética , DNA/genética , Dano ao DNA , Replicação do DNA , Camundongos , Camundongos Knockout , Micronúcleo Germinativo/genética , Micronúcleo Germinativo/imunologia , Mutação , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/metabolismo , Malformações do Sistema Nervoso/patologia , Ribonuclease H/genética , Ribonuclease H/metabolismo
2.
Cell Biol Int ; 32(3): 353-7, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18296078

RESUMO

Class II polypeptide release factor (eRF3), a ribosome and eRF1-dependent GTPase, is an important factor, which acts cooperatively with eRF1 to promote hydrolysis of the ester bond linking the polypeptide chain with the peptidyl site tRNA in process of termination of protein synthesis. We prepared antibodies against eRF3 of Euplotes octocarinatus, and performed localization studies by immunoelectron microscopy in the ciliate. Our results indicate that eRF3 is present both in the cytoplasm and the two types of nuclei of this organism. The functions of eRF3 in these nuclei were analyzed by RNA interference methods. The nuclei loose their shape in eRF3 gene-interfered Euplotes cells, suggesting that eRF3 is probably involved in the morphological organization of nuclei. This suggests that eRF3 is a multifunctional protein with roles additionals to its function in the process of termination of protein synthesis.


Assuntos
Núcleo Celular/enzimologia , Euplotes/enzimologia , Fatores de Terminação de Peptídeos/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Linhagem Celular , Núcleo Celular/ultraestrutura , Forma do Núcleo Celular , Citoplasma/enzimologia , Euplotes/ultraestrutura , Macronúcleo/enzimologia , Micronúcleo Germinativo/enzimologia , Microscopia Imunoeletrônica , Biossíntese de Proteínas , Proteínas de Protozoários/biossíntese , Interferência de RNA
3.
Eukaryot Cell ; 7(3): 471-82, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18178773

RESUMO

Newly synthesized histones are acetylated prior to their deposition into nucleosomes. Following nucleosome formation and positioning, they are rapidly deacetylated, an event that coincides with further maturation of the chromatin fiber. The histone deacetylases (HDACs) used for histone deposition and de novo chromatin formation are poorly understood. In the ciliate Tetrahymena thermophila, transcription-related deacetylation in the macronucleus is physically separated from deposition-related deacetylation in the micronucleus. This feature was utilized to identify an HDAC named Thd2, a class II HDAC that acts on newly synthesized histones to remove deposition-related acetyl moieties. The THD2 transcript is alternatively spliced, and the major form contains a putative inositol polyphosphate kinase (IPK) domain similar to Ipk2, an enzyme that promotes chromatin remodeling by SWI/SNF remodeling complexes. Cells lacking Thd2, which retain deposition-related acetyl moieties on new histones, exhibit chromatin and cytological phenotypes indicative of a role for Thd2 in chromatin maturation, including the proteolytic processing of histone H3.


Assuntos
Histona Desacetilases/metabolismo , Histonas/metabolismo , Tetrahymena thermophila/enzimologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Replicação do DNA , Macronúcleo/enzimologia , Micronúcleo Germinativo/enzimologia , Dados de Sequência Molecular , Nucleossomos/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Tetrahymena thermophila/metabolismo
4.
Eukaryot Cell ; 3(5): 1233-40, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15470252

RESUMO

The germ line micronucleus in Tetrahymena thermophila is transcriptionally silent in vegetatively growing cells. However, micronuclear transcription has been observed in the early ("crescent") stages of the sexual process, conjugation. This transcription is proposed to play a central role in identifying sites for subsequent genome rearrangements that accompany development of the somatic macronucleus from the micronucleus. RPB3 (cnjC), a gene encoding a protein homologous to the third largest subunit of RNA polymerase II (RNAP II), was previously reported to be expressed specifically during conjugation, suggesting a role in micronucleus-specific transcription. Rpb3p localized in the micronucleus only during the meiotic prophase, when micronuclear transcription occurs, and its intranuclear distribution is strikingly similar to that for previously described sites of micronuclear RNA synthesis. By contrast, Rpc5p, the homologous subunit shared by RNAPs I and III, was not detectable in the micronucleus at any stage of the life cycle. However, Rpb3p is not specific to the transcribing micronucleus. Like Rpc5p, it also localizes to macronuclei in all stages of the life cycle. Rpb3p is encoded by a unique, essential gene in Tetrahymena. Thus, RNAP II is associated with both somatic transcription and crescent transcription and probably has an important role in genome rearrangement.


Assuntos
RNA Polimerase II/metabolismo , Tetrahymena thermophila/enzimologia , Tetrahymena thermophila/genética , Animais , Sequência de Bases , DNA de Protozoário/genética , Rearranjo Gênico , Genes de Protozoários , Meiose , Micronúcleo Germinativo/enzimologia , Micronúcleo Germinativo/genética , Filogenia , Tetrahymena thermophila/crescimento & desenvolvimento , Transcrição Gênica
5.
Mol Cell ; 4(5): 695-704, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10619017

RESUMO

Ligation-mediated PCR was used to characterize intermediates in the fragmentation/de novo telomere addition process that occurs during sexual reproduction in the ciliate E. crassus. Fragmentation generates ends with 6-base, 3' overhangs that have 5'-phosphate and 3'-hydroxyl groups. These intermediates are detected only during the period of chromosome fragmentation. Fragmentation always occurs at a precise distance from a conserved sequence, the E-Cbs, indicating that it is a key cis-acting element in the process. The results also serve to identify the natural substrate for de novo telomere addition and indicate that telomerase recognizes, and compensates for, partial telomeric repeats at the ends of fragmentation intermediates. Similarities of the Euplotes fragmentation/telomere addition process to the movement of some non-long terminal repeat retrotransposons are discussed.


Assuntos
Quebra Cromossômica/genética , Cromossomos/metabolismo , Euplotes/crescimento & desenvolvimento , Euplotes/genética , Animais , Sequência de Bases , Núcleo Celular/enzimologia , Núcleo Celular/genética , Cromossomos/química , Cromossomos/genética , Sequência Conservada/genética , DNA Ligases/metabolismo , DNA de Protozoário/química , DNA de Protozoário/genética , DNA de Protozoário/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Euplotes/citologia , Euplotes/enzimologia , Evolução Molecular , Micronúcleo Germinativo/enzimologia , Micronúcleo Germinativo/genética , Modelos Genéticos , Reação em Cadeia da Polimerase/métodos , Sequências Reguladoras de Ácido Nucleico/genética , Reprodução , Retroelementos/genética , Telomerase/metabolismo , Telômero/química , Telômero/genética , Telômero/metabolismo
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