RESUMO
Dysfunction of the RNA-binding protein (RBP) FUS implicated in RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. Mutations affecting FUS nuclear localization can drive RNA splicing defects and stimulate the formation of non-amyloid inclusions in affected neurons. However, the mechanism by which FUS mutations contribute to the development of ALS remains uncertain. Here we describe a pattern of RNA splicing changes in the dynamics of the continuous proteinopathy induced by mislocalized FUS. We show that the decrease in intron retention of FUS-associated transcripts represents the hallmark of the pathogenesis of ALS and is the earliest molecular event in the course of progression of the disease. As FUS aggregation increases, the pattern of RNA splicing changes, becoming more complex, including a decrease in the inclusion of neuron-specific microexons and induction of cryptic exon splicing due to the sequestration of additional RBPs into FUS aggregates. Crucially, the identified features of the pathological splicing pattern are also observed in ALS patients in both sporadic and familial cases. Our data provide evidence that both a loss of nuclear FUS function due to mislocalization and the subsequent cytoplasmic aggregation of mutant protein lead to the disruption of RNA splicing in a multistep fashion during FUS aggregation.
Assuntos
Esclerose Lateral Amiotrófica , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Neurônios Motores/metabolismo , Mutação , Splicing de RNA/genética , Proteína FUS de Ligação a RNA/metabolismoRESUMO
Genomic and post-genomic editors based on CRISPR/Cas systems are widely used in basic research and applied sciences, including human gene therapy. Most genome editing tools are based on the CRISPR/Cas9 type IIA system from Streptococcus pyogenes. Unfortunately, a number of drawbacks have hindered its application in therapeutic approaches, the most serious of which is the relatively high level of off-targets. To overcome this obstacle, various high-fidelity Cas9 variants have been created. However, they show reduced on-target activity compared to wild-type Cas9 possibly due to increased sensitivity to eukaryotic chromatin. Here, we combined a rational approach with random mutagenesis to create a set of new Cas9 variants showing high specificity and increased activity in Saccharomyces cerevisiae yeast. Moreover, a novel mutation in the PAM (protospacer adjacent motif)-interacting Cas9 domain was found, which increases the on-target activity of high-fidelity Cas9 variants while retaining their high specificity. The obtained data suggest that this mutation acts by weakening the eukaryotic chromatin barrier for Cas9 and rearranging the RuvC active center. Improved Cas9 variants should further advance genome and post-genome editing technologies. KEY POINTS: ⢠D147Y and P411T mutations increase the activity of high-fidelity Cas9 variants. ⢠The new L1206P mutation further increases the activity of high-fidelity Cas9 variants. ⢠The L1206P mutation weakens the chromatin barrier for Cas9 editors.
Assuntos
Sistemas CRISPR-Cas , Humanos , Mutagênese , Edição de Genes , Cromatina , RNA Guia de Sistemas CRISPR-CasRESUMO
Distilled spirits production using Saccharomyces cerevisiae requires understanding of the mechanisms of yeast cell response to alcohol stress. Reportedly, specific mutations in genes of the ubiquitin-proteasome system, e.g., RPN4, may result in strains exhibiting hyper-resistance to different alcohols. To study the Rpn4-dependent yeast response to short-term ethanol exposure, we performed a comparative analysis of the wild-type (WT) strain, strain with RPN4 gene deletion (rpn4-Δ), and a mutant strain with decreased proteasome activity and consequent Rpn4 accumulation due to PRE1 deregulation (YPL). The stress resistance tests demonstrated an increased sensitivity of mutant strains to ethanol compared with WT. Comparative proteomics analysis revealed significant differences in molecular responses to ethanol between these strains. GO analysis of proteins upregulated in WT showed enrichments represented by oxidative and heat responses, protein folding/unfolding, and protein degradation. Enrichment of at least one of these responses was not observed in the mutant strains. Moreover, activity of autophagy was not increased in the RPN4 deletion strain upon ethanol stress which agrees with changes in mRNA levels of ATG7 and PRB1 genes of the autophagy system. Activity of the autophagic system was clearly induced and accompanied with PRB1 overexpression in the YPL strain upon ethanol stress. We demonstrated that Rpn4 stabilization contributes to the PRB1 upregulation. CRISPR-Cas9-mediated repression of PACE-core Rpn4 binding sites in the PRB1 promoter inhibits PRB1 induction in the YPL strain upon ethanol treatment and results in YPL hypersensitivity to ethanol. Our data suggest that Rpn4 affects the autophagic system activity upon ethanol stress through the PRB1 regulation. These findings can be a basis for creating genetically modified yeast strains resistant to high levels of alcohol, being further used for fermentation in ethanol production.
Assuntos
Autofagia/genética , Proteínas de Ligação a DNA/genética , Etanol/farmacologia , Complexo de Endopeptidases do Proteassoma , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Fatores de Transcrição/genética , Autofagia/efeitos dos fármacos , Endopeptidases/genética , Fermentação , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ativação TranscricionalRESUMO
Environmental and intracellular factors often damage DNA, but multiple DNA repair pathways maintain genome integrity. In yeast, the 26S proteasome and its transcriptional regulator and substrate Rpn4 are involved in DNA damage resistance. Paradoxically, while proteasome dysfunction may induce hyper-resistance to DNA-damaging agents, Rpn4 malfunction sensitizes yeasts to these agents. Previously, we proposed that proteasome inhibition causes Rpn4 stabilization followed by the upregulation of Rpn4-dependent DNA repair genes and pathways. Here, we aimed to elucidate the key Rpn4 targets responsible for DNA damage hyper-resistance in proteasome mutants. We impaired the Rpn4-mediated regulation of candidate genes using the CRISPR/Cas9 system and tested the sensitivity of mutant strains to 4-NQO, MMS and zeocin. We found that the separate or simultaneous deregulation of 19S or 20S proteasome subcomplexes induced MAG1, DDI1, RAD23 and RAD52 in an Rpn4-dependent manner. Deregulation of RAD23, DDI1 and RAD52 sensitized yeast to DNA damage. Genetic, epigenetic or dihydrocoumarin-mediated RAD52 repression restored the sensitivity of the proteasome mutants to DNA damage. Our results suggest that the Rpn4-mediated overexpression of DNA repair genes, especially RAD52, defines the DNA damage hyper-resistant phenotype of proteasome mutants. The developed yeast model is useful for characterizing drugs that reverse the DNA damage hyper-resistance phenotypes of cancers.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Sistemas CRISPR-Cas , Dano ao DNA , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Mutação , Proteína Rad52 de Recombinação e Reparo de DNA/antagonistas & inibidores , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genéticaRESUMO
The 26S proteasome participates in cell stress responses via its ability to degrade regulatory and damaged proteins. In yeast, mutations in the subunits of the 19S proteasome regulatory subcomplex cause hyper-resistance to 4-nitroquinoline-1-oxide (4-NQO), a chemical mutagen and carcinogen. These data suggest a negative role for the 19S proteasome complex in the cellular response to 4-NQO, although the underlying mechanism is not clear. We proposed that decreased 19S subcomplex activity leads to the stabilisation of Rpn4p, a transcription factor and proteasome substrate. In turn, stabilised Rpn4p may upregulate stress-responsive genes that participate in the response to 4-NQO-induced stress. To test our hypothesis, we impaired the expression of the RPT5 gene, which encodes the ATPase subunit of the 19S subcomplex, by mutating the Rpn4p binding site in its promoter. The mutant strain accumulates polyubiquitinated proteins-a hallmark of compromised proteasome function-and shows hyper-resistance to 4-NQO. We found several groups of genes that conferred resistance to 4-NQO-induced stress and were overexpressed due to the Rpn4p stabilisation and impaired 19S subcomplex function. The upregulated genes are involved in the oxidative and proteotoxic stress response pathways, multidrug resistance and biosynthesis of cysteine and methionine. Consistently, the mutant strain was hyper-resistant to oxidative stress. Our data imply that the ubiquitin-proteasome system may regulate the cellular response to 4-NQO at the transcriptional level.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/metabolismo , Quinolonas/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/biossíntese , Regulação para Cima , 4-Nitroquinolina-1-Óxido/metabolismo , Oxidantes/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Estresse FisiológicoRESUMO
The transcription factor ScRpn4 coordinates the expression of Saccharomyces cerevisiae proteasomal genes. ScRpn4 orthologues are found in a number of other Saccharomycetes yeasts. Their functions, however, have not yet been characterised experimentally in vivo . We expressed the Debaryomyces hansenii DEHA2D12848 gene encoding an ScRpn4 orthologue (DhRpn4), in an S. cerevisiae strain lacking RPN4 . We showed that DhRpn4 activates transcription of proteasomal genes using ScRpn4 binding site and provides resistance to various stresses. The 43-238 aa segment of DhRpn4 contains an unique portable transactivation domain. Similar to the ScRpn4 N-terminus, this domain lacks a compact structure Moreover, upon overexpression in D. hansenii , DhRpn4 upregulates protesomal genes. Thus, we show that DhRpn4 is the activator for proteasomal genes.
Assuntos
Regulação Fúngica da Expressão Gênica , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomycetales/enzimologia , Fatores de Transcrição/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Cyclin D1 is the activating subunit of the cell cycle kinases CDK4 and CDK6, and its dysregulation is a well-known oncogenic driver in many human cancers. The biological function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G2 and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unknown substrate of cyclin D1-CDK4/6. The phosphorylation of GTSE1 mediated by cyclin D1-CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 also during the G1 phase of the cell cycle. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1's role in cell cycle control and oncogenesis beyond RB phosphorylation.
RESUMO
Various external and internal factors damaging DNA constantly disrupt the stability of the genome. Cells use numerous dedicated DNA repair systems to detect damage and restore genomic integrity in a timely manner. Ribonucleotide reductase (RNR) is a key enzyme providing dNTPs for DNA repair. Molecular mechanisms of indirect regulation of yeast RNR activity are well understood, whereas little is known about its direct regulation. The study was aimed at elucidation of the proteasome-dependent mechanism of direct regulation of RNR subunits in Saccharomyces cerevisiae. Proteome analysis followed by Western blot, RT-PCR, and yeast plating analysis showed that upregulation of RNR by proteasome deregulation is associated with yeast hyper resistance to 4-nitroquinoline-1-oxide (4-NQO), a UV-mimetic DNA-damaging drug used in animal models to study oncogenesis. Inhibition of RNR or deletion of RNR regulatory proteins reverses the phenotype of yeast hyper resistance to 4-NQO. We have shown for the first time that the yeast Rnr1 subunit is a substrate of the proteasome, which suggests a common mechanism of RNR regulation in yeast and mammals.
RESUMO
Proteasomes are intracellular structures responsible for protein degradation. The 20S proteasome is a core catalytic element of the proteasome assembly. Variations of catalytic subunits generate different forms of 20S proteasomes including immunoproteasomes (iPs), which are present mostly in the immune cells. Certain cells of the immune system are primary targets of retroviruses. It has been shown that several viral proteins directly affect proteasome functionality, while inhibition of proteasome activity with broad specificity proteasome inhibitors stimulates viral transduction. Here we specifically addressed the role of the immunoproteasomes during early stages of viral transduction and investigated the effects of specific immunoproteasome inhibition and activation prior to infection using a panel of cell lines. Inhibition of iPs in hematopoietic cells with immunoproteasome-specific inhibitor ONX-0914 resulted in increased infection by VSV-G pseudotyped lentiviruses. Moreover, a tendency for increased infection of cloned cells with endogenously decreased proteasome activity was revealed. Conversely, activation of iPs by IFN-γ markedly reduced the viral infectivity, which was rescued upon simultaneous immunoproteasome inhibition. Our results indicate that immunoproteasome activity might be determinative for the cellular antiretroviral resistance at least for the cells with high iP content. Finally, therapeutic application of immunoproteasome inhibitors might promote retroviral infection of cells in vivo.
Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Lentivirus , Complexo de Endopeptidases do Proteassoma/imunologia , Antirretrovirais/farmacologia , Bortezomib/farmacologia , Linhagem Celular , Citocinas/metabolismo , Células HEK293 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Células HL-60 , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Retroviridae , Células THP-1 , Células U937RESUMO
The degradation of most intracellular proteins is a dynamic and tightly regulated process performed by proteasomes. To date, different forms of proteasomes have been identified. Currently the role of non-constitutive proteasomes (immunoproteasomes (iPs) and intermediate proteasomes (intPs)) has attracted special attention. Here, using a CRISPR-Cas9 nickase technology, four cell lines: histiocytic lymphoma, colorectal adenocarcinoma, cervix adenocarcinoma, and hepatocarcinoma were modified to express proteasomes with mCherry-tagged ß5i subunit, which is a catalytic subunit of iPs and intPs. Importantly, the expression of the chimeric gene in modified cells is under the control of endogenous regulatory mechanisms and is increased following IFN-γ and/or TNF-α stimulation. Fluorescent proteasomes retain catalytic activity and are distributed within the nucleus and cytoplasm. RNAseq reveals marginal differences in gene expression profiles between the modified and wild-type cell lines. Predominant metabolic pathways and patterns of expressed receptors were identified for each cell line. Using established cell lines, we demonstrated that anti-cancer drugs Ruxolitinib, Vincristine and Gefitinib stimulated the expression of ß5i-containing proteasomes, which might affect disease prognosis. Taken together, obtained cell lines can be used as a platform for real-time studies of immunoproteasome gene expression, localization of iPs and intPs, interaction of non-constitutive proteasomes with other proteins, proteasome trafficking and many other aspects of proteasome biology in living cells. Moreover, the established platform might be especially useful for fast and large-scale experiments intended to evaluate the effects of different conditions including treatment with various drugs and compounds on the proteasome pool.
Assuntos
Complexo de Endopeptidases do Proteassoma/imunologia , Subunidades Proteicas/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Fluorescência , Gefitinibe/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genoma Humano , Humanos , Interferon gama/farmacologia , Nitrilas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Vincristina/farmacologiaRESUMO
The marine yeast Debaryomyces hansenii is of high importance in the food, chemical, and medical industries. D. hansenii is also a popular model for studying molecular mechanisms of halo- and osmotolerance. The absence of genome editing technologies hampers D. hansenii research and limits its biotechnological application. We developed novel and efficient single- and dual-guide CRISPR systems for markerless genome editing of D. hansenii. The single-guide system allows high-efficiency (up to 95%) mutation of genes or regulatory elements. The dual-guide system is applicable for efficient deletion of genomic loci. We used these tools to study transcriptional regulation of the 26S proteasome, an ATP-dependent protease complex whose proper function is vital for all cells and organisms. We developed a genetic approach to control the activity of the 26S proteasome by deregulation of its essential subunits. The mutant strains were sensitive to geno- and proteotoxic stresses as well as high salinity and osmolarity, suggesting a contribution of the proteasome to the extremophilic properties of D. hansenii. The developed CRISPR systems allow efficient D. hansenii genome engineering, providing a genetic way to control proteasome activity, and should advance applications of this yeast.
Assuntos
Sistemas CRISPR-Cas , Debaryomyces/enzimologia , Debaryomyces/genética , Edição de Genes/métodos , Complexo de Endopeptidases do Proteassoma/genética , Saccharomyces cerevisiae/genética , Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Extremófilos/enzimologia , Extremófilos/genética , Regulação da Expressão Gênica , Genoma Fúngico , Organismos Geneticamente Modificados , Osmorregulação/genética , Estresse Oxidativo/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Salino/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaAssuntos
Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complexo de Endopeptidases do Proteassoma/biossíntese , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The 26S proteasome is an ATP-dependent multi-subunit protease complex and the major regulator of intracellular protein turnover and quality control. However, its role in the DNA damage response is controversial. We addressed this question in yeast by disrupting the transcriptional regulation of the PRE1 proteasomal gene. The mutant strain has decreased proteasome activity and is hyper-resistant to various DNA-damaging agents. We found that Rpn4-target genes MAG1, RAD23, and RAD52 are overexpressed in this strain due to Rpn4 stabilisation. These genes represent three different pathways of base excision, nucleotide excision and double strand break repair by homologous recombination (DSB-HR). Consistently, the proteasome mutant displays increased DSB-HR activity. Our data imply that the proteasome may have a negative role in DNA damage response.