RESUMO
Papillary thyroid carcinoma (PTC) is the most common histological category of thyroid cancer. In recent years, there has been an increasing number of studies on lncRNAs in PTC. Long intergenic non-protein coding RNA 887 (LINC00887) is a critical oncogene in developing other cancers. LINC00887 is upregulated in PTC samples but its role in PTC is currently unclear. This study aimed to investigate the impact the disruption of LINC00887 expression has on PTC progression. We performed a CRISPR/Cas9 strategy for the truncation of LINC00887 in BCPAP and TPC1 cell lines. Functional assays showed that LINC00887 knockdown in both TPC1 and BCPAP cells reduced cell proliferation, colony formation and migration, delayed the cell cycle, and increased apoptosis. These results strengthened the role of LINC00887 in cancer and showed for the first time that this lncRNA could be a potential oncogene in PTC, acting as a tumor promoter. Modulation of the immune system may be one of the etiopathogenic mechanisms of LINC00887 in PTC, as shown by the observed influence of this lncRNA on PD-L1 expression. In addition, the biological pathways of LINC00887 identified to date, such as EMT, the Wnt/ß-catenin signaling pathway or the FRMD6-Hippo signaling pathway may also be relevant regulatory mechanisms operating in PTC.
Assuntos
Carcinoma Papilar , RNA Longo não Codificante , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , RNA Longo não Codificante/genética , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Regulação para Cima/genéticaRESUMO
Rad50 contains a conserved Zn(2+) coordination domain (the Rad50 hook) that functions as a homodimerization interface. Hook ablation phenocopies Rad50 deficiency in all respects. Here, we focused on rad50 mutations flanking the Zn(2+)-coordinating hook cysteines. These mutants impaired hook-mediated dimerization, but recombination between sister chromatids was largely unaffected. This may reflect that cohesin-mediated sister chromatid interactions are sufficient for double-strand break repair. However, Mre11 complex functions specified by the globular domain, including Tel1 (ATM) activation, nonhomologous end joining, and DNA double-strand break end resection were affected, suggesting that dimerization exerts a broad influence on Mre11 complex function. These phenotypes were suppressed by mutations within the coiled-coil and globular ATPase domains, suggesting a model in which conformational changes in the hook and globular domains are transmitted via the extended coils of Rad50. We propose that transmission of spatial information in this manner underlies the regulation of Mre11 complex functions.
Assuntos
Cromátides/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Cromossomos Fúngicos/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Mutação , Fenótipo , Conformação Proteica , Multimerização Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Thyroid carcinoma (TC) can be classified as medullary (MTC) and non-medullary (NMTC). While most TCs are sporadic, familial forms of MTC and NMTC also exist (less than 1% and 3-9% of all TC cases, respectively). Germline mutations in RET are found in more than 95% of familial MTC, whereas familial NMTC shows a high degree of genetic heterogeneity. Herein, we aimed to identify susceptibility genes for familial NMTC and non-RET MTC by whole exome sequencing in 58 individuals belonging to 18 Spanish families with these carcinomas. After data analysis, 53 rare candidate segregating variants were identified in 12 of the families, 7 of them located in previously TC-associated genes. Although no common mutated genes were detected, biological processes regulating functions such as cell proliferation, differentiation, survival and adhesion were enriched. The reported functions of the identified genes together with pathogenicity and structural predictions, reinforced the candidacy of 36 of them, suggesting new loci related to TC and novel genotype-phenotype correlations. Therefore, our strategy provides clues to possible molecular mechanisms underlying familial forms of MTC and NMTC. These new molecular findings and clinical data of patients may be helpful for the early detection, development of tailored therapies and optimizing patient management.
Assuntos
Carcinoma , Neoplasias da Glândula Tireoide , Humanos , Sequenciamento do Exoma , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Mutação em Linhagem GerminativaRESUMO
The THO complex is a key factor in co-transcriptional formation of export-competent messenger ribonucleoprotein particles, yet its structure and mechanism of chromatin recruitment remain unknown. In yeast, this complex has been described as a heterotetramer (Tho2, Hpr1, Mft1, and Thp2) that interacts with Tex1 and mRNA export factors Sub2 and Yra1 to form the TRanscription EXport (TREX) complex. In this study, we purified yeast THO and found Tex1 to be part of its core. We determined the three-dimensional structures of five-subunit THO complex by electron microscopy and located the positions of Tex1, Hpr1, and Tho2 C-terminus using various labelling techniques. In the case of Tex1, a ß-propeller protein, we have generated an atomic model which docks into the corresponding part of the THO complex envelope. Furthermore, we show that THO directly interacts with nucleic acids through the unfolded C-terminal region of Tho2, whose removal reduces THO recruitment to active chromatin leading to mRNA biogenesis defects. In summary, this study describes the THO architecture, the structural basis for its chromatin targeting, and highlights the importance of unfolded regions of eukaryotic proteins.
Assuntos
Ácidos Nucleicos/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
To clarify the role of a number of mRNA processing factors in transcription elongation, we developed an in vivo assay for direct analysis of elongation on chromatin. The assay relies on two substrates containing two G-less cassettes separated by either a long and GC-rich or a short and GC-poor DNA sequence (G-less-based run-on (GLRO) assay). We demonstrate that PAF, THSC/TREX-2, SAGA, the exosome component Rrp6 and two subunits of cleavage factor IA (Rna14 and Rna15) are required for efficient transcription elongation, in contrast to some results obtained using other assays. Next, we undertook a mutant screen and found out that the Nup84 nucleoporin complex is also required for transcription elongation, as confirmed by the GLRO assay and RNA polymerase II chromatin immunoprecipitations. Therefore, in addition to showing that the GLRO assay is a sensitive and reliable method for the analysis of elongation in vivo, this study provides evidence for a new role of the Nup84 complex and a number of mRNA processing factors in transcription elongation that supports a connection of pre-mRNA processing and nuclear export with transcription elongation.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Transcrição Gênica , Imunoprecipitação da Cromatina , Elementos de DNA Transponíveis , Técnicas de Inativação de Genes , Biologia Molecular/métodos , Mutagênese Insercional , Complexo de Proteínas Formadoras de Poros Nucleares/genética , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Yeast rad3-102, a mutant of the TFIIH complex involved in nucleotide excision repair (NER) and transcription, can perform NER initial steps but not late steps of postincision gap filing. Because removal of early-acting NER proteins prevents rad3-102 deleterious action, we used this feature to explore if chaperones act in early NER. We found that the cochaperone Ydj1 is required for NER and that Ydj1 guarantees TFIIH stoichiometry. Importantly, in the absence of Ydj1, the roles of TFIIH in transcription and transactivation, the ability to activate transcription by nuclear receptors in response to hormones, are strongly impaired. We propose that TFIIH constitutes a multitarget complex for Ydj1, as six of the seven TFIIH core components contain biologically relevant Ydj1- binding motives. Our results provide evidence for a role of chaperones in NER and transcription, with implications in cancer and TFIIH-associated syndromes.
Assuntos
Reparo do DNA , Proteínas de Choque Térmico HSP40/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fator de Transcrição TFIIH/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIH/química , Ativação Transcricional/genéticaRESUMO
RNA plays an indispensable role in mammalian cell functions. Cas13, a class of RNA-guided ribonuclease, is a flexible tool for modifying and regulating coding and non-coding RNAs, with enormous potential for creating new cell functions. However, the lack of control over Cas13 activity has limited its cell engineering capability. Here, we present the CRISTAL (Control of RNA with Inducible SpliT CAs13 Orthologs and Exogenous Ligands) platform. CRISTAL is powered by a collection (10 total) of orthogonal split inducible Cas13 effectors that can be turned ON or OFF via small molecules in multiple cell types, providing precise temporal control. Also, we engineer Cas13 logic circuits that can respond to endogenous signaling and exogenous small molecule inputs. Furthermore, the orthogonality, low leakiness, and high dynamic range of our inducible Cas13d and Cas13b enable the design and construction of a robust incoherent feedforward loop, leading to near-perfect and tunable adaptation response. Finally, using our inducible Cas13 effectors, we achieve simultaneous multiplexed control of multiple genes in vitro and in mice. Together, our CRISTAL design represents a powerful platform for precisely regulating RNA dynamics to advance cell engineering and elucidate RNA biology.
Assuntos
Sistemas CRISPR-Cas , RNA , Animais , Camundongos , RNA/genética , Mamíferos/genéticaRESUMO
Hirschsprung's disease (HSCR) is a rare developmental disorder in which enteric ganglia are missing along a portion of the intestine. HSCR has a complex inheritance, with RET as the major disease-causing gene. However, the pathogenesis of HSCR is still not completely understood. Therefore, we applied a computational approach based on multi-omics network characterization and clustering analysis for HSCR-related gene/miRNA identification and biomarker discovery. Protein-protein interaction (PPI) and miRNA-target interaction (MTI) networks were analyzed by DPClusO and BiClusO, respectively, and finally, the biomarker potential of miRNAs was computationally screened by miRNA-BD. In this study, a total of 55 significant gene-disease modules were identified, allowing us to propose 178 new HSCR candidate genes and two biological pathways. Moreover, we identified 12 key miRNAs with biomarker potential among 137 predicted HSCR-associated miRNAs. Functional analysis of new candidates showed that enrichment terms related to gene ontology (GO) and pathways were associated with HSCR. In conclusion, this approach has allowed us to decipher new clues of the etiopathogenesis of HSCR, although molecular experiments are further needed for clinical validations.
Assuntos
Doença de Hirschsprung , MicroRNAs , Humanos , Doença de Hirschsprung/genética , Multiômica , MicroRNAs/genética , Biologia Computacional , BiomarcadoresRESUMO
RNA plays an indispensable role in mammalian cell functions. Cas13, a class of RNA-guided ribonuclease, is a flexible tool for modifying and regulating coding and non-coding RNAs, with enormous potential for creating new cell functions. However, the lack of control over Cas13 activity has limited its cell engineering capability. Here, we present the CRISTAL ( C ontrol of R NA with Inducible S pli T C A s13 Orthologs and Exogenous L igands) platform. CRISTAL is powered by a collection (10 total) of orthogonal split inducible Cas13s that can be turned ON or OFF via small molecules in multiple cell types, providing precise temporal control. Also, we engineered Cas13 logic circuits that can respond to endogenous signaling and exogenous small molecule inputs. Furthermore, the orthogonality, low leakiness, and high dynamic range of our inducible Cas13d and Cas13b enable the design and construction of a robust incoherent feedforward loop, leading to near-perfect and tunable adaptation response. Finally, using our inducible Cas13s, we achieve simultaneous multiplexed control of multiple genes in vitro and in mice. Together, our CRISTAL design represents a powerful platform for precisely regulating RNA dynamics to advance cell engineering and elucidate RNA biology.
RESUMO
RNA polymerases frequently deal with a number of obstacles during transcription elongation that need to be removed for transcription resumption. One important type of hindrance consists of DNA lesions, which are removed by transcription-coupled repair (TC-NER), a specific sub-pathway of nucleotide excision repair. To improve our knowledge of transcription elongation and its coupling to TC-NER, we used the yeast library of non-essential knock-out mutations to screen for genes conferring resistance to the transcription-elongation inhibitor mycophenolic acid and the DNA-damaging agent 4-nitroquinoline-N-oxide. Our data provide evidence that subunits of the SAGA and Ccr4-Not complexes, Mediator, Bre1, Bur2, and Fun12 affect transcription elongation to different extents. Given the dependency of TC-NER on RNA Polymerase II transcription and the fact that the few proteins known to be involved in TC-NER are related to transcription, we performed an in-depth TC-NER analysis of a selection of mutants. We found that mutants of the PAF and Ccr4-Not complexes are impaired in TC-NER. This study provides evidence that PAF and Ccr4-Not are required for efficient TC-NER in yeast, unraveling a novel function for these transcription complexes and opening new perspectives for the understanding of TC-NER and its functional interconnection with transcription elongation.
Assuntos
Reparo do DNA/fisiologia , Genoma Fúngico , Ribonucleases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA Fúngico/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , RNA Mensageiro/metabolismo , Ribonucleases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
Small molecule-inducible gene circuits are some of the most important tools in biology because they provide a convenient way to exert precise regulation of biological systems. These systems typically are designed to govern gene activation, repression, or disruption at multiple levels, such as through genome modification, transcription, translation, or post-translational regulation of protein activity. Due to their importance, many new systems have been created in the past few years to address different needs or afford orthogonality. They can be broadly characterized based on the inducer used, the mode of regulation, and the effector protein enabling the regulation. Furthermore, each synthetic circuit has varying performance metrics and design considerations. Here, we provide a concise comparison of recently developed tools and recommend standardized metrics for evaluating their performance and potential as biological interrogators or therapeutics.
Assuntos
Regulação da Expressão Gênica , Redes Reguladoras de Genes , Animais , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Genoma , Proteínas/genética , Biologia Sintética , Mamíferos/genéticaRESUMO
Homologous recombination (HR) is the major mechanism used to repair double-strand breaks (DSBs) that result from replication, but a study of repair of DSBs specifically induced during S-phase is lacking. Using an inverted-repeat assay in which a DSB is generated by the encountering of the replication fork with nicks, we can physically detect repair by sister-chromatid recombination (SCR) and intra-chromatid break-induced replication (IC-BIR). As expected, both events depend on Rad52, but, in contrast to previous data, both require Rad59, suggesting a prominent role of Rad59 in repair of replication-born DSBs. In the absence of Rad51, SCR is severely affected while IC-BIR increases, a phenotype that is also observed in the absence of Rad54 but not of its paralog Rdh54/Tid1. These data are consistent with SCR occurring by Rad51-dependent mechanisms assisted by Rad54, and indicate that in the absence of strand exchange-dependent SCR, breaks can be channeled to IC-BIR, which works efficiently in the absence of Rad51. Our study provides molecular evidence for inversions between repeats occurring by BIR followed by single-strand annealing (SSA) in the absence of strand exchange.
Assuntos
Cromátides/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Recombinação Genética , Saccharomyces cerevisiae/genética , DNA Helicases , Enzimas Reparadoras do DNA , DNA Topoisomerases , Proteínas de Ligação a DNA/fisiologia , Rad51 Recombinase/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Troca de Cromátide IrmãRESUMO
Sarcomas are malignant tumors accounting for a high percentage of cancer morbidity and mortality in children and young adults. Surgery and radiation therapy are the accepted treatments for most sarcomas; however, patients with metastatic disease are treated with systemic chemotherapy. Many tumors display marginal levels of chemoresponsiveness, and new treatment approaches are needed. MAP17 is a small non-glycosylated membrane protein overexpressed in carcinomas. The levels of MAP17 could be used as a prognostic marker to predict the response to bortezomib in hematological malignancies and in breast tumors. Therefore, we analyzed the expression of this oncogene in sarcomas and its relationship with clinico-pathological features, as well as tested whether it can be used as a new biomarker to predict the therapeutic response to bortezomib and new therapies for sarcomas. We found that the levels of MAP17 were related to clinical features and poor survival in a cohort of 69 patients with different sarcoma types, not being restricted to any special subtype of tumor. MAP17 expression is associated with poor overall survival (p<0.001) and worse disease-free survival (p=0.002). Cell lines with high levels of MAP17 show a better response to bortezomib in vitro. Furthermore, patient-derived xenografts (PDX) with high levels of MAP17 respond to bortezomib in vivo. Our results showed that this response is due to the lower levels of NFκB and autophagy activation. Therefore, we suggest that MAP17 is a new biomarker to predict the efficacy of bortezomib as a new therapy for sarcomas.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/análise , Bortezomib/uso terapêutico , Proteínas de Membrana/biossíntese , Adolescente , Adulto , Idoso , Animais , Área Sob a Curva , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos , Pessoa de Meia-Idade , Prognóstico , Curva ROC , Sarcoma/tratamento farmacológico , Sarcoma/metabolismo , Sensibilidade e Especificidade , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
Most spontaneous DNA double-strand breaks (DSBs) arise during replication and are repaired by homologous recombination (HR) with the sister chromatid. Many proteins participate in HR, but it is often difficult to determine their in vivo functions due to the existence of alternative pathways. Here we take advantage of an in vivo assay to assess repair of a specific replication-born DSB by sister chromatid recombination (SCR). We analyzed the functional relevance of four structure-selective endonucleases (SSEs), Yen1, Mus81-Mms4, Slx1-Slx4, and Rad1, on SCR in Saccharomyces cerevisiae. Physical and genetic analyses showed that ablation of any of these SSEs leads to a specific SCR decrease that is not observed in general HR. Our work suggests that Yen1, Mus81-Mms4, Slx4, and Rad1, but not Slx1, function independently in the cleavage of intercrossed DNA structures to reconstitute broken replication forks via HR with the sister chromatid. These unique effects, which have not been detected in other studies unless double mutant combinations were used, indicate the formation of distinct alternatives for the repair of replication-born DSBs that require specific SSEs.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA Fúngico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Troca de Cromátide Irmã , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Endonucleases Flap/genética , Endonucleases Flap/metabolismo , Resolvases de Junção Holliday/genética , Resolvases de Junção Holliday/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Formation of a ribonucleoprotein particle (mRNP) competent for export requires the coupling of transcription with mRNA processing and RNA export. A key link between these processes is provided by the THO complex. To progress in our understanding of this coupling, we have performed a search for suppressors of the transcription defect caused by the hpr1Δ mutation. This has permitted us to identify mutations in the genes for the RNA polymerase II mediator component Med10, the Sch9 protein kinase, and the Ypr045c protein. We report a role in transcription elongation for Ypr045c (Thp3) and the Csn12 component of the COP9 signalosome. Thp3 and Csn12 form a complex that is recruited to transcribed genes. Their mutations suppress the gene expression defects of THO complex mutants involved in mRNP biogenesis and export and show defects in mRNA accumulation. Transcription elongation impairment of thp3Δ mutants is shown by in vivo transcript run-on analysis performed in G-less systems. Thp3-Csn12 establishes a novel link between transcription and mRNA processing that opens new perspectives on our understanding of gene expression and reveals novel functions for a component of the COP9 signalosome. Thp3-Csn12 also copurifies with ribosomal proteins, which opens the possibility that it has other functions in addition to transcription.
Assuntos
Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Complexo do Signalossomo COP9 , DNA Fúngico/genética , Genes Fúngicos , Genoma Fúngico , Complexo Mediador/genética , Complexo Mediador/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fases de Leitura Aberta , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Processamento Pós-Transcricional do RNA , Supressão Genética , Transcrição GênicaRESUMO
Gene expression in eukaryotes is an essential process that includes transcription, RNA processing, and export. One important player in this interface is the poly(A)(+)-RNA-binding protein Nab2, which regulates the mRNA poly(A)(+)-tail length and export. Here we show that Nab2 has additional roles during mRNA transcription, tRNA metabolism, and ribosomal subunit export. Nab2 is associated with the entire open reading frame of actively transcribed RNA polymerase (RNAP) II and III genes. As a consequence, nab2 mutations confer translation defects that are detected by polysome profiling. Genome-wide analysis of expression of a conditional degron nab2 mutant shows that the role of Nab2 in RNAPII transcription and RNAPIII metabolism is direct. Taken together, our results identify novel functions for Nab2 in transcription and metabolism of most types of RNAs, indicating that Nab2 function is more ubiquitous than previously anticipated, and that it is a central player in the general and coordinated control of gene expression from transcription to translation.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas de Transporte Nucleocitoplasmático/genética , Poli A/metabolismo , Polirribossomos/metabolismo , RNA Polimerase III/genética , RNA Polimerase II/genética , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Northern Blotting , Núcleo Celular/genética , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Deleção de Genes , Perfilação da Expressão Gênica , Proteínas de Transporte Nucleocitoplasmático/deficiência , Organismos Geneticamente Modificados , Poli A/genética , Polirribossomos/genética , Análise Serial de Proteínas , Biossíntese de Proteínas , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcrição GênicaRESUMO
The Mre11 complex (Mre11, Rad50 and Xrs2 in Saccharomyces cerevisiae) influences diverse functions in the DNA damage response. The complex comprises the globular DNA-binding domain and the Rad50 hook domain, which are linked by a long and extended Rad50 coiled-coil domain. In this study, we constructed rad50 alleles encoding truncations of the coiled-coil domain to determine which Mre11 complex functions required the full length of the coils. These mutations abolished telomere maintenance and meiotic double-strand break (DSB) formation, and severely impaired homologous recombination, indicating a requirement for long-range action. Nonhomologous end joining, which is probably mediated by the globular domain of the Mre11 complex, was also severely impaired by alteration of the coiled-coil and hook domains, providing the first evidence of their influence on this process. These data show that functions of Mre11 complex are integrated by the coiled coils of Rad50.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Endodesoxirribonucleases/fisiologia , Exodesoxirribonucleases/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Cromátides/metabolismo , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Mutação , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and plays a role in preventing the transcription-associated genetic instability. THO is composed of Tho2, Hpr1, Mft1 and Thp2 subunits, which associate with the Sub2-Yra1 export factors and Tex1 to form the TREX complex. To compare the functional relevance of the different THO/TREX subunits, we determined the effect of their null mutations on mRNA accumulation and recombination. Unexpectedly, we noticed that a full deletion of HPR1, hpr1DeltaK, conferred stronger hyper-recombination phenotype and gene expression defects than did hpr1DeltaH, the allele encoding a C-terminal truncated protein which was used in most previous studies. We show that tho2Delta and, to a lesser extent, hpr1DeltaK are the THO mutations with the highest impact on all phenotypes, and that sub2Delta shows a similar transcription-dependent hyper-recombination phenotype and in vivo transcription impairment as hpr1DeltaK and tho2Delta. Recombination and transcription analyses indicate that THO/TREX mutants share a moderate but significant effect on gene conversion and ectopic recombination, as well as transcription impairment of even short and low GC-content genes. Our data provide new information on the relevance of these proteins in mRNP biogenesis and in the maintenance of genomic integrity.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Alelos , Northern Blotting , Proteínas de Transporte/metabolismo , Proteínas de Transporte/fisiologia , Cromossomos Fúngicos/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Modelos Genéticos , Mutação , Fenótipo , Plasmídeos/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Subunidades Proteicas/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação Genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Transcrição GênicaRESUMO
The eukaryotic THO/TREX complex, involved in mRNP biogenesis, plays a key role in the maintenance of genome integrity in yeast. mRNA export factors such as Thp1-Sac3 also affect genome integrity, but their mutations have other phenotypes different from those of THO/TREX. Sus1 is a novel component of SAGA transcription factor that also associates with Thp1-Sac3, but little is known about its effect on genome instability and transcription. Here we show that Thp1, Sac3, and Sus1 form a functional unit with a role in mRNP biogenesis and maintenance of genome integrity that is independent of SAGA. Importantly, the effects of ribozyme-containing transcription units, RNase H, and the action of human activation-induced cytidine deaminase on transcription and genome instability are consistent with the possibility that R-loops are formed in Thp1-Sac3-Sus1-Cdc31 as in THO mutants. Our data reveal that Thp1-Sac3-Sus1-Cdc31, together with THO/TREX, define a specific pathway connecting transcription elongation with export via an RNA-dependent dynamic process that provides a feedback mechanism for the control of transcription and the preservation of genetic integrity of transcribed DNA regions.