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2.
Cell Rep ; 42(11): 113325, 2023 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-37889751

RESUMO

The RNA exosome is a versatile ribonuclease. In the nucleoplasm of mammalian cells, it is assisted by its adaptors the nuclear exosome targeting (NEXT) complex and the poly(A) exosome targeting (PAXT) connection. Via its association with the ARS2 and ZC3H18 proteins, NEXT/exosome is recruited to capped and short unadenylated transcripts. Conversely, PAXT/exosome is considered to target longer and adenylated substrates via their poly(A) tails. Here, mutational analysis of the core PAXT component ZFC3H1 uncovers a separate branch of the PAXT pathway, which targets short adenylated RNAs and relies on a direct ARS2-ZFC3H1 interaction. We further demonstrate that similar acidic-rich short linear motifs of ZFC3H1 and ZC3H18 compete for a common ARS2 epitope. Consequently, while promoting NEXT function, ZC3H18 antagonizes PAXT activity. We suggest that this organization of RNA decay complexes provides co-activation of NEXT and PAXT at loci with abundant production of short exosome substrates.


Assuntos
RNA Nuclear , Proteínas de Ligação a RNA , Animais , Núcleo Celular/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Mamíferos , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Nuclear/genética , Proteínas de Ligação a RNA/genética
3.
Mol Cell ; 83(13): 2240-2257.e6, 2023 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-37329882

RESUMO

The RNA-binding ARS2 protein is centrally involved in both early RNA polymerase II (RNAPII) transcription termination and transcript decay. Despite its essential nature, the mechanisms by which ARS2 enacts these functions have remained unclear. Here, we show that a conserved basic domain of ARS2 binds a corresponding acidic-rich, short linear motif (SLiM) in the transcription restriction factor ZC3H4. This interaction recruits ZC3H4 to chromatin to elicit RNAPII termination, independent of other early termination pathways defined by the cleavage and polyadenylation (CPA) and Integrator (INT) complexes. We find that ZC3H4, in turn, forms a direct connection to the nuclear exosome targeting (NEXT) complex, hereby facilitating rapid degradation of the nascent RNA. Hence, ARS2 instructs the coupled transcription termination and degradation of the transcript onto which it is bound. This contrasts with ARS2 function at CPA-instructed termination sites where the protein exclusively partakes in RNA suppression via post-transcriptional decay.


Assuntos
Proteínas Nucleares , Transcrição Gênica , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA
4.
RNA ; 26(12): 1935-1956, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32963109

RESUMO

The NineTeen Complex (NTC), also known as pre-mRNA-processing factor 19 (Prp19) complex, regulates distinct spliceosome conformational changes necessary for splicing. During Drosophila midblastula transition, splicing is particularly sensitive to mutations in NTC-subunit Fandango, which suggests differential requirements of NTC during development. We show that NTC-subunit Salsa, the Drosophila ortholog of human RNA helicase Aquarius, is rate-limiting for splicing of a subset of small first introns during oogenesis, including the first intron of gurken Germline depletion of Salsa and splice site mutations within gurken first intron impair both adult female fertility and oocyte dorsal-ventral patterning, due to an abnormal expression of Gurken. Supporting causality, the fertility and dorsal-ventral patterning defects observed after Salsa depletion could be suppressed by the expression of a gurken construct without its first intron. Altogether, our results suggest that one of the key rate-limiting functions of Salsa during oogenesis is to ensure the correct expression and efficient splicing of the first intron of gurken mRNA. Retention of gurken first intron compromises the function of this gene most likely because it undermines the correct structure and function of the transcript 5'UTR.


Assuntos
Padronização Corporal/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Íntrons/genética , Splicing de RNA , Fator de Crescimento Transformador alfa/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Feminino , Infertilidade Feminina/etiologia , Infertilidade Feminina/metabolismo , Infertilidade Feminina/patologia , Spliceossomos/genética , Spliceossomos/metabolismo , Fator de Crescimento Transformador alfa/genética
5.
Curr Biol ; 28(17): 2837-2844.e3, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30122528

RESUMO

The fidelity of mitosis depends on cohesive forces that keep sister chromatids together. This is mediated by cohesin that embraces sister chromatid fibers from the time of their replication until the subsequent mitosis [1-3]. Cleavage of cohesin marks anaphase onset, where single chromatids are dragged to the poles by the mitotic spindle [4-6]. Cohesin cleavage should only occur when all chromosomes are properly bio-oriented to ensure equal genome distribution and prevent random chromosome segregation. Unscheduled loss of sister chromatid cohesion is prevented by a safeguard mechanism known as the spindle assembly checkpoint (SAC) [7, 8]. To identify specific conditions capable of restoring defects associated with cohesion loss, we screened for genes whose depletion modulates Drosophila wing development when sister chromatid cohesion is impaired. Cohesion deficiency was induced by knockdown of the acetyltransferase separation anxiety (San)/Naa50, a cohesin complex stabilizer [9-12]. Several genes whose function impacts wing development upon cohesion loss were identified. Surprisingly, knockdown of key SAC proteins, Mad2 and Mps1, suppressed developmental defects associated with San depletion. SAC impairment upon cohesin removal, triggered by San depletion or artificial removal of the cohesin complex, prevented extensive genome shuffling, reduced segregation defects, and restored cell survival. This counterintuitive phenotypic suppression was caused by an intrinsic bias for efficient chromosome biorientation at mitotic entry, coupled with slow engagement of error-correction reactions. Thus, in contrast to SAC's role as a safeguard mechanism for mitotic fidelity, removal of this checkpoint alleviates mitotic errors when sister chromatid cohesion is compromised.


Assuntos
Drosophila melanogaster/citologia , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Mitose/fisiologia , Troca de Cromátide Irmã/fisiologia , Animais
6.
Sci Rep ; 6: 39118, 2016 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-27996020

RESUMO

The gene separation anxiety (san) encodes Naa50/San, a N-terminal acetyltransferase required for chromosome segregation during mitosis. Although highly conserved among higher eukaryotes, the mitotic function of this enzyme is still poorly understood. Naa50/San was originally proposed to be required for centromeric sister chromatid cohesion in Drosophila and human cells, yet, more recently, it was also suggested to be a negative regulator of microtubule polymerization through internal acetylation of beta Tubulin. We used genetic and biochemical approaches to clarify the function of Naa50/San during development. Our work suggests that Naa50/San is required during tissue proliferation for the correct interaction between the cohesin subunits Scc1 and Smc3. Our results also suggest a working model where Naa50/San N-terminally acetylates the nascent Scc1 polypeptide, and that this co-translational modification is subsequently required for the establishment and/or maintenance of sister chromatid cohesion.


Assuntos
Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Acetilação , Adenosina Trifosfatases/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Segregação de Cromossomos , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo
7.
Sci Rep ; 6: 21304, 2016 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-26861501

RESUMO

Protein N-terminal acetylation is an ancient and ubiquitous co-translational modification catalyzed by a highly conserved family of N-terminal acetyltransferases (NATs). Prokaryotes have at least 3 NATs, whereas humans have six distinct but highly conserved NATs, suggesting an increase in regulatory complexity of this modification during eukaryotic evolution. Despite this, and against our initial expectations, we determined that NAT diversification did not occur in the eukaryotes, as all six major human NATs were most likely present in the Last Eukaryotic Common Ancestor (LECA). Furthermore, we also observed that some NATs were actually secondarily lost during evolution of major eukaryotic lineages; therefore, the increased complexity of the higher eukaryotic proteome occurred without a concomitant diversification of NAT complexes.


Assuntos
Arabidopsis/enzimologia , Evolução Biológica , Drosophila melanogaster/enzimologia , Células Eucarióticas/enzimologia , Acetiltransferases N-Terminal/genética , Saccharomyces cerevisiae/enzimologia , Acetilação , Sequência de Aminoácidos , Animais , Arabidopsis/metabolismo , Drosophila melanogaster/metabolismo , Células Eucarióticas/metabolismo , Variação Genética , Humanos , Proteoma/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência
8.
Leuk Lymphoma ; 56(11): 3168-82, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25772975

RESUMO

The present work aimed to investigate the anticancer in vitro activity of two plants commonly used in traditional Indian medicine: Zingiber officinale Roscoe and Nerium oleander L. The extracts of these plants were tested in vitro on several human leukemic cell lines, K562, THP-1, MOLT-4 and Jurkat. Cell growth inhibition was observed for both plant extracts with 50% inhibitory concentration (IC50) values ranging between 1 and 28 µg/mL using SRB (sulphorodamine B) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assays. Enhanced cell growth inhibition was observed when the extracts were combined with imatinib. Exposed cells showed cell cycle arrest, DNA damage and cytochrome c release, indicating that the mechanism of cytotoxicity could be via mitochondrial mediated apoptotic pathways. Combination of the extracts of these plants with standard cancer treatment may be a way of enhancing responses. Clinical studies in patients with chronic myeloid leukemia are planned at our center.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Índia , Concentração Inibidora 50 , Leucemia , Extratos Vegetais/química
9.
Leuk Lymphoma ; 55(11): 2614-9, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24446903

RESUMO

The present study looked at the correlation between mean trough Imatinib plasma levels and molecular response in 131 CML patients on imatinib. Patients receiving Glivec versus generic Imatinib were also compared. A ROC curve was constructed to estimate a threshold level that correlates with a favourable response. Patients were grouped into Responders (bcr/abl ration by RQ-PCR less than 1) and Non Responders (ration ≥ 1). The mean trough imatinib plasma level in the responders was significantly higher than in the non responders (p = 0.001). The area under ROC curve was 0.733, with best sensitivity (51.85%) and specificity (89.42%) at a plasma threshold of 0.988 g/ml [1.675 M]. Levels in the patients on Glivec versus generic drug (p > 0.05) were comparable. Trough Imatinib plasma levels may be a marker for suboptimal response and may identify patients in whom increase of drug dose or change in therapy may be indicated.


Assuntos
Benzamidas/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Adolescente , Adulto , Antineoplásicos/efeitos adversos , Antineoplásicos/sangue , Antineoplásicos/uso terapêutico , Benzamidas/efeitos adversos , Benzamidas/sangue , Diarreia/induzido quimicamente , Fadiga/induzido quimicamente , Feminino , Proteínas de Fusão bcr-abl/genética , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Masculino , Pessoa de Meia-Idade , Cãibra Muscular/induzido quimicamente , Náusea/induzido quimicamente , Avaliação de Resultados em Cuidados de Saúde/métodos , Piperazinas/efeitos adversos , Piperazinas/sangue , Pirimidinas/efeitos adversos , Pirimidinas/sangue , Indução de Remissão , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto Jovem
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