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1.
Curr Opin Struct Biol ; 84: 102766, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38181687

RESUMO

RNA polymerase II (Pol II) transcription is regulated by many elongation factors. Among these factors, TFIIF, PAF-RTF1, ELL and Elongin stimulate mRNA chain elongation by Pol II. Cryo-EM structures of Pol II complexes with these elongation factors now reveal some general principles on how elongation factors bind Pol II and how they stimulate transcription. All four elongation factors contact Pol II at domains external 2 and protrusion, whereas TFIIF and ELL additionally bind the Pol II lobe. All factors apparently stabilize cleft-flanking elements, whereas RTF1 and Elongin additionally approach the active site with a latch element and may influence catalysis or translocation. Due to the shared binding sites on Pol II, factor binding is mutually exclusive, and thus it remains to be studied what determines which elongation factors bind at a certain gene and under which condition.


Assuntos
RNA Polimerase II , Fatores de Transcrição TFII , RNA Polimerase II/química , Elonguina/genética , Elonguina/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Transcrição TFII/química , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismo , Sítios de Ligação , Transcrição Gênica
2.
Sci Adv ; 9(47): eadj1261, 2023 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-37992162

RESUMO

The biological role of the repetitive DNA sequences in the human genome remains an outstanding question. Recent long-read human genome assemblies have allowed us to identify a function for one of these repetitive regions. We have uncovered a tandem array of conserved primate-specific retrogenes encoding the protein Elongin A3 (ELOA3), a homolog of the RNA polymerase II (RNAPII) elongation factor Elongin A (ELOA). Our genomic analysis shows that the ELOA3 gene cluster is conserved among primates and the number of ELOA3 gene repeats is variable in the human population and across primate species. Moreover, the gene cluster has undergone concerted evolution and homogenization within primates. Our biochemical studies show that ELOA3 functions as a promoter-associated RNAPII pause-release elongation factor with distinct biochemical and functional features from its ancestral homolog, ELOA. We propose that the ELOA3 gene cluster has evolved to fulfil a transcriptional regulatory function unique to the primate lineage that can be targeted to regulate cellular hyperproliferation.


Assuntos
Fatores de Alongamento de Peptídeos , RNA Polimerase II , Animais , Humanos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fatores de Alongamento de Peptídeos/genética , Primatas/genética , Elonguina/genética , Família Multigênica , Sequências de Repetição em Tandem/genética
3.
Nat Struct Mol Biol ; 30(12): 1925-1935, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37932450

RESUMO

Elongin is a heterotrimeric elongation factor for RNA polymerase (Pol) II transcription that is conserved among metazoa. Here, we report three cryo-EM structures of human Elongin bound to transcribing Pol II. The structures show that Elongin subunit ELOA binds the RPB2 side of Pol II and anchors the ELOB-ELOC subunit heterodimer. ELOA contains a 'latch' that binds between the end of the Pol II bridge helix and funnel helices, thereby inducing a conformational change near the polymerase active center. The latch is required for the elongation-stimulatory activity of Elongin, but not for Pol II binding, indicating that Elongin functions by allosterically regulating the conformational mobility of the polymerase active center. Elongin binding to Pol II is incompatible with association of the super elongation complex, PAF1 complex and RTF1, which also contain an elongation-stimulatory latch element.


Assuntos
RNA Polimerase II , Fatores de Transcrição , Humanos , Elonguina/genética , Elonguina/metabolismo , Fatores de Transcrição/metabolismo , RNA Polimerase II/metabolismo , Núcleo Celular/metabolismo , Transcrição Gênica
4.
Cell Chem Biol ; 30(7): 766-779.e11, 2023 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-37354906

RESUMO

Inhibition of protein-protein interactions (PPIs) via designed peptides is an effective strategy to perturb their biological functions. The Elongin BC heterodimer (ELOB/C) binds to a BC-box motif and is essential for cancer cell growth. Here, we report a peptide that mimics the high-affinity BC-box of the PRC2-associated protein EPOP. This peptide tightly binds to the ELOB/C dimer (kD = 0.46 ± 0.02 nM) and blocks the association of ELOB/C with its interaction partners, both in vitro and in the cellular environment. Cancer cells treated with our peptide inhibitor showed decreased cell viability, increased apoptosis, and perturbed gene expression. Therefore, our work proposes that blocking the BC-box-binding pocket of ELOB/C is a feasible strategy to impair its function and inhibit cancer cell growth. Our peptide inhibitor promises novel mechanistic insights into the biological function of the ELOB/C dimer and offers a starting point for therapeutics linked to ELOB/C dysfunction.


Assuntos
Neoplasias , Fatores de Transcrição , Elonguina/metabolismo , Fatores de Transcrição/metabolismo , Ligação Proteica , Peptídeos/farmacologia , Peptídeos/metabolismo , Apoptose , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias/tratamento farmacológico
5.
Mod Pathol ; 36(8): 100194, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37088333

RESUMO

Approximately 70% of clear cell renal cell carcinoma (ccRCC) is characterized by the biallelic inactivation of von Hippel-Lindau (VHL) on chromosome 3p. ELOC-mutated (Elongin C-mutated) renal cell carcinoma containing biallelic ELOC inactivations with chromosome 8q deletions is considered a novel subtype of renal cancer possessing a morphologic overlap with ccRCC, renal cell carcinoma (RCC) with fibromyomatous stroma exhibiting Tuberous Sclerosis Complex (TSC)/mammalian Target of Rapamycin (mTOR) mutations, and clear cell papillary tumor. However, the frequency and consequences of ELOC alterations in wild-type VHL and mutated VHL RCC are unclear. In this study, we characterize 123 renal tumors with clear cell morphology and known VHL mutation status to assess the morphologic and molecular consequences of ELOC inactivation. Using OncoScan and whole-exome sequencing, we identify 18 ELOC-deleted RCCs, 3 of which contain ELOC mutations resulting in the biallelic inactivation of ELOC. Biallelic ELOC and biallelic VHL aberrations were mutually exclusive; however, 2 ELOC-mutated RCCs showed monoallelic VHL alterations. Furthermore, no mutations in TSC1, TSC2, or mTOR were identified in ELOC-mutated RCC with biallelic ELOC inactivation. Using High Ambiguity Driven biomolecular DOCKing, we report a novel ELOC variant containing a duplication event disrupting ELOC-VHL interaction alongside the frequently seen Y79C alteration. Using hyper reaction monitoring mass spectrometry, we show RCCs with biallelic ELOC alterations have significantly reduced ELOC expression but similar carbonic anhydrase 9 and vascular endothelial growth factor A expression compared with classical ccRCC with biallelic VHL inactivation. The absence of biallelic VHL and TSC1, TSC2, or mTOR inactivation in RCC with biallelic ELOC inactivation (ELOC mutation in combination with ELOC deletions on chromosome 8q) supports the notion of a novel, molecularly defined tumor entity.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/patologia , Fator A de Crescimento do Endotélio Vascular , Elonguina/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Neoplasias Renais/genética , Neoplasias Renais/patologia , Serina-Treonina Quinases TOR
6.
Mol Cancer Res ; 21(5): 428-443, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-36753617

RESUMO

High expression of the receptor tyrosine kinase AXL is implicated in epithelial-to-mesenchymal transition, cancer progression, and therapy resistance. For example, AXL is abundant in BRAF mutant melanomas progressing on targeted BRAF/MEK inhibition. Therefore, AXL is thought to represent an attractive therapeutic target. This notwithstanding, little is known about the mechanisms governing expression of AXL. Here, we describe a FACS-based whole-genome-wide CRISPR-Cas9 screen to uncover regulators of AXL expression. We identified several genes, inactivation of which led to increased AXL expression. Most remarkable was the identification of five components that associate with the Elongin BC heterodimer. Elongin B/C engage in multiple protein-protein interactions, including the transcription factor complex subunit Elongin A, the von Hippel-Lindau (VHL) tumor suppressor protein, and members of the SOCS-box protein family. The screen identified ELOB, ELOC, SOCS5, UBE2F, and RNF7, each of which we demonstrate to serve as an inhibitor of AXL expression. Although the AXL promoter contains hypoxia response elements and Elongin B/C are found in the VHL complex, Elongin B/C unexpectedly regulate AXL independently of hypoxia. Instead, we demonstrate that the Elongin BC complex interacts with AXL through ELOB, and contributes to proteasomal AXL turnover. RNA-sequencing and IHC analyses of melanoma patient-derived xenografts and clinical samples revealed a negative association between Elongin B/C and dedifferentiation. Together, the Elongin BC complex regulates AXL and marks a differentiated melanoma phenotype. IMPLICATIONS: This study identifies the Elongin BC complex as a key regulator of AXL expression and marker of melanoma differentiation.


Assuntos
Melanoma , Ubiquitina-Proteína Ligases , Humanos , Elonguina , Melanoma/genética , Complexo de Endopeptidases do Proteassoma , Proteínas Proto-Oncogênicas B-raf , Fatores de Transcrição/genética , Proteína Supressora de Tumor Von Hippel-Lindau , Receptores Proteína Tirosina Quinases/metabolismo
7.
Int J Mol Sci ; 24(4)2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36835292

RESUMO

The basic helix-loop-helix factors play a central role in neuronal differentiation and nervous system development, which involve the Notch and signal transducer and activator of transcription (STAT)/small mother against decapentaplegic signaling pathways. Neural stem cells differentiate into three nervous system lineages, and the suppressor of cytokine signaling (SOCS) and von Hippel-Lindau (VHL) proteins are involved in this neuronal differentiation. The SOCS and VHL proteins both contain homologous structures comprising the BC-box motif. SOCSs recruit Elongin C, Elongin B, Cullin5(Cul5), and Rbx2, whereas VHL recruits Elongin C, Elongin B, Cul2, and Rbx1. SOCSs form SBC-Cul5/E3 complexes, and VHL forms a VBC-Cul2/E3 complex. These complexes degrade the target protein and suppress its downstream transduction pathway by acting as E3 ligases via the ubiquitin-proteasome system. The Janus kinase (JAK) is the main target protein of the E3 ligase SBC-Cul5, whereas hypoxia-inducible factor is the primary target protein of the E3 ligase VBC-Cul2; nonetheless, VBC-Cul2 also targets the JAK. SOCSs not only act on the ubiquitin-proteasome system but also act directly on JAKs to suppress the Janus kinase-signal transduction and activator of transcription (JAK-STAT) pathway. Both SOCS and VHL are expressed in the nervous system, predominantly in brain neurons in the embryonic stage. Both SOCS and VHL induce neuronal differentiation. SOCS is involved in differentiation into neurons, whereas VHL is involved in differentiation into neurons and oligodendrocytes; both proteins promote neurite outgrowth. It has also been suggested that the inactivation of these proteins may lead to the development of nervous system malignancies and that these proteins may function as tumor suppressors. The mechanism of action of SOCS and VHL involved in neuronal differentiation and nervous system development is thought to be mediated through the inhibition of downstream signaling pathways, JAK-STAT, and hypoxia-inducible factor-vascular endothelial growth factor pathways. In addition, because SOCS and VHL promote nerve regeneration, they are expected to be applied in neuronal regenerative medicine for traumatic brain injury and stroke.


Assuntos
Neurogênese , Proteínas Supressoras da Sinalização de Citocina , Fator A de Crescimento do Endotélio Vascular , Proteína Supressora de Tumor Von Hippel-Lindau , Humanos , Diferenciação Celular , Proteínas Culina/metabolismo , Elonguina/metabolismo , Janus Quinases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina , Ubiquitina-Proteína Ligases/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo
8.
Biochem Soc Trans ; 51(1): 125-135, 2023 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-36651856

RESUMO

Interaction scaffolds that selectively recognize disordered protein strongly shape protein interactomes. An important scaffold of this type that contributes to transcription is the TFIIS N-terminal domain (TND). The TND is a five-helical bundle that has no known enzymatic activity, but instead selectively reads intrinsically disordered sequences of other proteins. Here, we review the structural and functional properties of TNDs and their cognate disordered ligands known as TND-interacting motifs (TIMs). TNDs or TIMs are found in prominent members of the transcription machinery, including TFIIS, super elongation complex, SWI/SNF, Mediator, IWS1, SPT6, PP1-PNUTS phosphatase, elongin, H3K36me3 readers, the transcription factor MYC, and others. We also review how the TND interactome contributes to the regulation of transcription. Because the TND is the most significantly enriched fold among transcription elongation regulators, TND- and TIM-driven interactions have widespread roles in the regulation of many transcriptional processes.


Assuntos
Fatores de Transcrição , Fatores de Elongação da Transcrição , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Elonguina/metabolismo , Regulação da Expressão Gênica
9.
Cell Death Differ ; 30(1): 137-151, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-35995846

RESUMO

Radioresistance is a principal culprit for the failure of radiotherapy in hepatocellular carcinoma (HCC). Insights on the regulation genes of radioresistance and underlying mechanisms in HCC are awaiting for profound investigation. In this study, the suppressor of cytokine signaling 2 (SOCS2) were screened out by RNA-seq and bioinformatics analyses as a potential prognosis predictor of HCC radiotherapy and then were determined to promote radiosensitivity in HCC both in vivo or in vitro. Meanwhile, the measurements of ferroptosis negative regulatory proteins of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4), intracellular lipid peroxidation and Fe2+ concentration suggested that a high level of ferroptosis contributed to the radiosensitization of HCC. Moreover, SOCS2 and SLC7A11 were expressed oppositely in HCC clinical tissues and tumour xenografts with different radiosensitivities. Mechanistically, the N-terminal domain of SLC7A11 was specifically recognized by the SH2-structural domain of SOCS2. While the L162 and C166 of SOCS2-BOX region could bind elongin B/C compound to co-form a SOCS2/elongin B/C complex to recruit ubiquitin molecules. Herein, SOCS2 served as a bridge to transfer the attached ubiquitin to SLC7A11 and promoted K48-linked polyubiquitination degradation of SLC7A11, which ultimately led to the onset of ferroptosis and radiosensitization of HCC. In conclusion, it was demonstrated for the first time that high-expressed SOCS2 was one of the biomarkers predicting radiosensitivity of HCC by advancing the ubiquitination degradation of SLC7A11 and promoting ferroptosis, which indicates that targeting SOCS2 may enhance the efficiency of HCC radiotherapy and improve the prognosis of patients.


Assuntos
Carcinoma Hepatocelular , Ferroptose , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/radioterapia , Carcinoma Hepatocelular/metabolismo , Elonguina/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Sistema y+ de Transporte de Aminoácidos/genética , Sistema y+ de Transporte de Aminoácidos/metabolismo
10.
Mol Cancer ; 21(1): 210, 2022 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-36376892

RESUMO

BACKGROUND: Long noncoding RNAs (lncRNAs) have driven research focused on their effects as oncogenes or tumor suppressors involved in carcinogenesis. However, the functions and mechanisms of most lncRNAs in colorectal cancer (CRC) remain unclear. METHODS: The expression of DLGAP1-AS2 was assessed by quantitative RT-PCR in multiple CRC cohorts. The impacts of DLGAP1-AS2 on CRC growth and metastasis were evaluated by a series of in vitro and in vivo assays. Furthermore, the underlying mechanism of DLGAP1-AS2 in CRC was revealed by RNA pull down, RNA immunoprecipitation, RNA sequencing, luciferase assays, chromatin immunoprecipitation, and rescue experiments. RESULTS: We discovered that DLGAP1-AS2 promoted CRC tumorigenesis and metastasis by physically interacting with Elongin A (ELOA) and inhibiting its protein stability by promoting tripartite motif containing 21 (Trim21)-mediated ubiquitination modification and degradation of ELOA. In particular, we revealed that DLGAP1-AS2 decreases phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) expression by inhibiting ELOA-mediated transcriptional activating of LHPP and thus blocking LHPP-dependent suppression of the AKT signaling pathway. In addition, we also demonstrated that DLGAP1-AS2 was bound and stabilized by cleavage and polyadenylation specificity factor (CPSF2) and cleavage stimulation factor (CSTF3). CONCLUSIONS: The discovery of DLGAP1-AS2, a promising prognostic biomarker, reveals a new dimension into the molecular pathogenesis of CRC and provides a prospective treatment target for this disease.


Assuntos
Neoplasias Colorretais , MicroRNAs , RNA Longo não Codificante , Humanos , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Transformação Celular Neoplásica/genética , Fator de Especificidade de Clivagem e Poliadenilação/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Neoplasias Colorretais/patologia , Elonguina/genética , Elonguina/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo
11.
Proc Natl Acad Sci U S A ; 119(40): e2207332119, 2022 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-36161924

RESUMO

Rpb1, the largest subunit of RNA polymerase II (RNAPII), is rapidly polyubiquitinated and degraded in response to DNA damage; this process is considered to be a "mechanism of last resort'' employed by cells. The underlying mechanism of this process remains elusive. Here, we uncovered a previously uncharacterized multistep pathway in which the polymerase-associated factor 1 (Paf1) complex (PAF1C, composed of the subunits Ctr9, Paf1, Leo1, Cdc73, and Rtf1) is involved in regulating the RNAPII pool by stimulating Elongin-Cullin E3 ligase complex-mediated Rpb1 polyubiquitination and subsequent degradation by the proteasome following DNA damage. Mechanistically, Spt5 is dephosphorylated following DNA damage, thereby weakening the interaction between the Rtf1 subunit and Spt5, which might be a key step in initiating Rpb1 degradation. Next, Rad26 is loaded onto stalled RNAPII to replace the Spt4/Spt5 complex in an RNAPII-dependent manner and, in turn, recruits more PAF1C to DNA lesions via the binding of Rad26 to the Leo1 subunit. Importantly, the PAF1C, assembled in a Ctr9-mediated manner, coordinates with Rad26 to localize the Elongin-Cullin complex on stalled RNAPII, thereby inducing RNAPII removal, in which the heterodimer Paf1/Leo1 and the subunit Cdc73 play important roles. Together, our results clearly revealed a new role of the intact PAF1C in regulating the RNAPII pool in response to DNA damage.


Assuntos
Proteínas Culina , Dano ao DNA , Elonguina , Proteínas Nucleares , RNA Polimerase II , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Proteínas Culina/metabolismo , Elonguina/genética , Elonguina/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/metabolismo
12.
Pathol Res Pract ; 235: 153960, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35653922

RESUMO

OBJECTIVE: This study aimed to investigate the clinicopathological and molecular characteristics of ELOC(TCEB1)-mutant renal cell carcinoma. METHODS: Sanger sequencing was used to assess 32 cases originally diagnosed as clear cell renal cell carcinoma with CK7 positive and/or fibromyomatous stroma. Of these, 4 patients with ELOC(TCEB1) gene mutation were screened, and their clinicopathological data were collected for histomorphological observation, immunohistochemical staining, and follow-up, and relevant pieces of literature were reviewed. RESULTS: The 4 patients with ELOC(TCEB1) mutations were all males and aged between 57 and 64 years (median age: 59 years old). The tumor was located in the renal cortex, with a diameter of 2-3.5 cm. The cross-section was grayish-yellow and grayish brown, solid and nodular, and clearly demarcated from the surrounding tissues. Of the 4 patients, 3 harbored a thick fibrous pseudocapsule rich in smooth muscle and were separated from the surrounding normal renal tissue, and 2 of them showed focal invasion into the pseudocapsule, whereas 1 patient had no capsule but had focal invasion into the surrounding renal parenchyma. The tumor tissues mainly exhibited elongated or branched aciniform or tubular structures, commonly accompanied by interspersed small cystic and focal clustered short papillary structures. The cytoplasm of the tumor cells was rich and lightly stained, and the nuclear grading ranged from 1 to 2. All patients showed loose edema in the stroma, and 2 patients showed a small number of interspersed smooth muscle bundles. All 4 patients showed EMA, CA9, AMACR, and TCEB1 expression, and TCEB1 was mainly located in the nucleus. Vimentin, CK7, and CD10 expressions were observed in most cases; CD117, TFE3, HMB45, and melanA were not expressed in all tumors; the expression rate of Ki67 was 3%- 8%. All 4 patients had a point mutation in ELOC(TCEB1) Y79C. The patients were followed up for 24-93 months (mean 49 months), and all of them survived to date without recurrence or metastasis. CONCLUSION: ELOC(TCEB1)-mutant renal cell carcinoma is a rare type of renal cell carcinoma, which tends to occur in middle-aged and elderly men. The main characteristics of this tumor are the branching alveolar or tubular structure with clustered short papillae, presence of fibromyomatous stroma, and the expression of CK7, CA9, CD10, and AMACR. Positive TCEB1 nuclear staining may be an important marker and the Sanger sequencing method is helpful for the diagnosis of this type of RCC. Most patients harbor tumors exhibiting low nuclear grade and inert clinical behavior, and a few tumors exhibit high nuclear grade and aggressive characteristics.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/patologia , Elonguina/genética , Humanos , Imuno-Histoquímica , Neoplasias Renais/patologia , Erros Inatos do Metabolismo Lipídico , Masculino , Pessoa de Meia-Idade , Neprilisina , Doenças do Sistema Nervoso , Racemases e Epimerases/deficiência
13.
Hum Mol Genet ; 31(16): 2728-2737, 2022 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-35323939

RESUMO

Around 95% of patients with clinical features that meet the diagnostic criteria for von Hippel-Lindau disease (VHL) have a detectable inactivating germline variant in VHL. The VHL protein (pVHL) functions as part of the E3 ubiquitin ligase complex comprising pVHL, elongin C, elongin B, cullin 2 and ring box 1 (VCB-CR complex), which plays a key role in oxygen sensing and degradation of hypoxia-inducible factors. To date, only variants in VHL have been shown to cause VHL disease. We undertook trio analysis by whole-exome sequencing in a proband with VHL disease but without a detectable VHL mutation. Molecular studies were also performed on paired DNA extracted from the proband's kidney tumour and blood and bioinformatics analysis of sporadic renal cell carcinoma (RCC) dataset was undertaken. A de novo pathogenic variant in ELOC NM_005648.4(ELOC):c.236A>G (p.Tyr79Cys) gene was identified in the proband. ELOC encodes elongin C, a key component [C] of the VCB-CR complex. The p.Tyr79Cys substitution is a mutational hotspot in sporadic VHL-competent RCC and has previously been shown to mimic the effects of pVHL deficiency on hypoxic signalling. Analysis of an RCC from the proband showed similar findings to that in somatically ELOC-mutated RCC (expression of hypoxia-responsive proteins, no somatic VHL variants and chromosome 8 loss). These findings are consistent with pathogenic ELOC variants being a novel cause for VHL disease and suggest that genetic testing for ELOC variants should be performed in individuals with suspected VHL disease with no detectable VHL variant.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Doença de von Hippel-Lindau , Carcinoma de Células Renais/genética , Elonguina/genética , Humanos , Hipóxia , Neoplasias Renais/genética , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Doença de von Hippel-Lindau/genética
14.
EMBO J ; 40(23): e108271, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34605059

RESUMO

Mutations in the gene encoding the CDKL5 kinase are among the most common genetic causes of childhood epilepsy and can also give rise to the severe neurodevelopmental condition CDD (CDKL5 deficiency disorder). Despite its importance for human health, the phosphorylation targets and cellular roles of CDKL5 are poorly understood, especially in the cell nucleus. Here, we report that CDKL5 is recruited to sites of DNA damage in actively transcribed regions of the nucleus. A quantitative phosphoproteomic screen for nuclear CDKL5 substrates reveals a network of transcriptional regulators including Elongin A (ELOA), phosphorylated on a specific CDKL5 consensus motif. Recruitment of CDKL5 and ELOA to damaged DNA, and subsequent phosphorylation of ELOA, requires both active transcription and the synthesis of poly(ADP-ribose) (PAR), to which CDKL5 can bind. Critically, CDKL5 kinase activity is essential for the transcriptional silencing of genes induced by DNA double-strand breaks. Thus, CDKL5 is a DNA damage-sensing, PAR-controlled transcriptional modulator, a finding with implications for understanding the molecular basis of CDKL5-related diseases.


Assuntos
Quebras de DNA de Cadeia Dupla , Dano ao DNA , Elonguina/metabolismo , Neurônios/patologia , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ativação Transcricional , Elonguina/genética , Síndromes Epilépticas/genética , Síndromes Epilépticas/metabolismo , Síndromes Epilépticas/patologia , Humanos , Mutação , Neurônios/metabolismo , Fosfoproteínas/genética , Fosforilação , Poli Adenosina Difosfato Ribose/metabolismo , Proteínas Serina-Treonina Quinases/genética , Espasmos Infantis/genética , Espasmos Infantis/metabolismo , Espasmos Infantis/patologia
15.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34544872

RESUMO

The bZIP transcription factor ATF6α is a master regulator of endoplasmic reticulum (ER) stress response genes. In this report, we identify the multifunctional RNA polymerase II transcription factor Elongin as a cofactor for ATF6α-dependent transcription activation. Biochemical studies reveal that Elongin functions at least in part by facilitating ATF6α-dependent loading of Mediator at the promoters and enhancers of ER stress response genes. Depletion of Elongin from cells leads to impaired transcription of ER stress response genes and to defects in the recruitment of Mediator and its CDK8 kinase subunit. Taken together, these findings bring to light a role for Elongin as a loading factor for Mediator during the ER stress response.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Elonguina/metabolismo , Estresse do Retículo Endoplasmático , Regulação da Expressão Gênica , Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Fator 6 Ativador da Transcrição/genética , Animais , Elonguina/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Células HeLa , Humanos , Complexo Mediador/genética , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Ratos , Transdução de Sinais , Ativação Transcricional
16.
Nat Commun ; 12(1): 4961, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400640

RESUMO

Esophageal cancer (EC) is a type of aggressive cancer without clinically relevant molecular subtypes, hindering the development of effective strategies for treatment. To define molecular subtypes of EC, we perform mass spectrometry-based proteomic and phosphoproteomics profiling of EC tumors and adjacent non-tumor tissues, revealing a catalog of proteins and phosphosites that are dysregulated in ECs. The EC cohort is stratified into two molecular subtypes-S1 and S2-based on proteomic analysis, with the S2 subtype characterized by the upregulation of spliceosomal and ribosomal proteins, and being more aggressive. Moreover, we identify a subtype signature composed of ELOA and SCAF4, and construct a subtype diagnostic and prognostic model. Potential drugs are predicted for treating patients of S2 subtype, and three candidate drugs are validated to inhibit EC. Taken together, our proteomic analysis define molecular subtypes of EC, thus providing a potential therapeutic outlook for improving disease outcomes in patients with EC.


Assuntos
Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Espectrometria de Massas/métodos , Proteômica , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Ciclo Celular , Estudos de Coortes , Elonguina/genética , Elonguina/metabolismo , Humanos , Prognóstico , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo
17.
J Biol Chem ; 297(1): 100862, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34116057

RESUMO

The Elongin complex was originally identified as an RNA polymerase II (RNAPII) elongation factor and subsequently as the substrate recognition component of a Cullin-RING E3 ubiquitin ligase. More recent evidence indicates that the Elongin ubiquitin ligase assembles with the Cockayne syndrome B helicase (CSB) in response to DNA damage and can target stalled polymerases for ubiquitylation and removal from the genome. In this report, we present evidence that the CSB-Elongin ubiquitin ligase pathway has roles beyond the DNA damage response in the activation of RNAPII-mediated transcription. We observed that assembly of the CSB-Elongin ubiquitin ligase is induced not just by DNA damage, but also by a variety of signals that activate RNAPII-mediated transcription, including endoplasmic reticulum (ER) stress, amino acid starvation, retinoic acid, glucocorticoids, and doxycycline treatment of cells carrying several copies of a doxycycline-inducible reporter. Using glucocorticoid receptor (GR)-regulated genes as a model, we showed that glucocorticoid-induced transcription is accompanied by rapid recruitment of CSB and the Elongin ubiquitin ligase to target genes in a step that depends upon the presence of transcribing RNAPII on those genes. Consistent with the idea that the CSB-Elongin pathway plays a direct role in GR-regulated transcription, mouse cells lacking the Elongin subunit Elongin A exhibit delays in both RNAPII accumulation on and dismissal from target genes following glucocorticoid addition and withdrawal, respectively. Taken together, our findings bring to light a new role for the CSB-Elongin pathway in RNAPII-mediated transcription.


Assuntos
DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Elonguina/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Polimerase II/genética , Ubiquitina-Proteína Ligases/genética , Animais , Síndrome de Cockayne/enzimologia , Síndrome de Cockayne/genética , DNA Helicases/química , DNA Helicases/ultraestrutura , Reparo do DNA/genética , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/ultraestrutura , Elonguina/química , Elonguina/ultraestrutura , Humanos , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/ultraestrutura , RNA Polimerase II/química , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Ubiquitina/química , Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/ultraestrutura , Ubiquitinação/genética
18.
Oncogene ; 40(30): 4919-4929, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34163032

RESUMO

Previous study demonstrated that most long non-coding RNAs (lncRNAs) function as competing endogenous RNAs or molecular sponges to negatively modulate miRNA and regulate tumor development. However, the molecular mechanisms of lncRNAs in cancer are not fully understood. Our study describes the role of the lncRNA SPRY4 intronic transcript 1 (SPRY4-IT1) in cancer metastasis by mechanisms related to Staufen1 (STAU1)-mediated mRNA decay (SMD). Briefly, we found that, high SPRY4-IT1 expression was associated with aggressiveness and poor outcome in human colorectal, breast and ovarian cancer tissues. In addition, functional assays revealed that SPRY4-IT1 significantly promoted colorectal, breast and ovarian cancer metastasis in vitro and in vivo. Mechanistically, microarray analyses identified several differentially-expressed genes upon SPRY4-IT1 overexpression in HCT 116 colorectal cancer cells. Among them, the 3'-UTR of transcription elongation factor B subunit 1 (TCEB1) mRNA can base-pair with the Alu element in the 3'-UTR of SPRY4-IT1. Moreover, SPRY4-IT1 was found to bind STAU1, promote STAU1 recruitment to the 3'-UTR of TCEB1 mRNA, and affect TCEB1 mRNA stability and expression, resulting in hypoxia-inducible factor 1α (HIF-1α) upregulation, and thereby affecting cancer cell metastasis. In addition, STAU1 depletion abrogated TCEB1 SMD and alleviated the pro-metastatic effect of SPRY4-IT1 overexpression. Significantly, we revealed that SPRY4-IT1 is also transactivated by NF-κB/p65, which activates SPRY4-IT1 to inhibit TCEB1 expression, and subsequently upregulate HIF-1α. In conclusion, our results highlight a novel mechanism of cytoplasmic lncRNA SPRY4-IT1 in which SPRY4-IT1 affecting TCEB1 mRNA stability via STAU1-mediated degradation during cancer metastasis.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Elonguina/genética , NF-kappa B/metabolismo , Estabilidade de RNA , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Elementos Alu , Sítios de Ligação , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Interferência de RNA , Transdução de Sinais
19.
J Biol Chem ; 296: 100202, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33334895

RESUMO

Elongin A (EloA) is an essential transcription factor that stimulates the rate of RNA polymerase II (Pol II) transcription elongation in vitro. However, its role as a transcription factor in vivo has remained underexplored. Here we show that in mouse embryonic stem cells, EloA localizes to both thousands of Pol II transcribed genes with preference for transcription start site and promoter regions and a large number of active enhancers across the genome. EloA deletion results in accumulation of transcripts from a subset of enhancers and their adjacent genes. Notably, EloA does not substantially enhance the elongation rate of Pol II in vivo. We also show that EloA localizes to the nucleoli and associates with RNA polymerase I transcribed ribosomal RNA gene, Rn45s. EloA is a highly disordered protein, which we demonstrate forms phase-separated condensates in vitro, and truncation mutations in the intrinsically disordered regions (IDR) of EloA interfere with its targeting and localization to the nucleoli. We conclude that EloA broadly associates with transcribed regions, tunes RNA Pol II transcription levels via impacts on enhancer RNA synthesis, and interacts with the rRNA producing/processing machinery in the nucleolus. Our work opens new avenues for further investigation of the role of this functionally multifaceted transcription factor in enhancer and ribosomal RNA biology.


Assuntos
Elonguina/metabolismo , Elementos Facilitadores Genéticos , Células-Tronco Embrionárias Murinas/metabolismo , RNA/genética , Ativação Transcricional , Animais , Linhagem Celular , Elonguina/genética , Deleção de Genes , Camundongos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Sítio de Iniciação de Transcrição
20.
Virology ; 554: 17-27, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33333348

RESUMO

The family of human APOBEC3 (A3) restriction factors is formed by seven different proteins, A3A-D and A3F-H. Among these A3s, A3B harbors strong restriction activity against several retroviruses, such as SIV, and MLV. How lentiviruses and other retroviruses, prevalent in many primate species, counteract A3B is poorly understood. In this study, we found that A3B strongly inhibited SIVmac and HIV-2 infectivity, which was antagonized by their Vif proteins. Both SIVmac and HIV-2 Vifs diminished the protein level of A3B in viral producer cells, and hindered A3B incorporation into viral particles. We observed that HIV-2 Vif binds A3B and induces its degradation by assembly of an A3-Vif-CUL5-ElonginB/C E3-ligase complex. A3B and HIV-2 Vif localize and interact in the nucleus. In addition, we also found that the accessory protein Bet of prototype foamy virus (PFV) significantly antagonized the anti-SIVmac activity of A3B. Like Vif, Bet prevented the incorporation of A3B into viral particles. However, in contrast to Vif Bet did not induce the degradation of A3B. Rather, Bet binds A3B to block formation of high molecular weight A3B complexes and induces A3B cytoplasmic trapping. In summary, these findings indicate that A3B is recognized by diverse retroviruses and counteracted by virus-specific pathways that could be targeted to inhibit A3B mutating activity in cancers.


Assuntos
Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/metabolismo , HIV-2/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas dos Retroviridae/metabolismo , Spumavirus/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Elonguina/genética , Elonguina/metabolismo , Produtos do Gene vif/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Vírus da Imunodeficiência Símia/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Vírion/metabolismo
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