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1.
Medicine (Baltimore) ; 98(39): e17337, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31574871

RESUMO

RATIONALE: Diamond-Blackfan anemia (DBA) is a rare inherited marrow disorder, characterized by erythrocyte aplasia and is associated with congenital anomalies and a susceptibility to cancer. Although congenital abnormalities have been observed in ∼50% of DBA patients, the occurrence of an associated congenital diaphragmatic hernia (CDH) has rarely been reported. PATIENT CONCERNS: A 19-month-old male child was referred to our pediatric hematology-oncology outpatient clinic with anemic appearance. He presented to us with recurrent anemia, short stature, and developmental delay. DIAGNOSIS: On bone marrow examination, only erythropoietic cells were markedly decreased in number, whereas other cell lines were unaffected. An abdominal computed tomography scan revealed a Bochdalek type of CDH. A genetic analysis revealed heterozygous mutation of RPS19; therefore, he was diagnosed as having DBA with CDH. INTERVENTIONS: The patient received an initial packed red blood cell transfusion, followed by an administration of oral prednisone. OUTCOMES: The patient is maintained on oral prednisone administered at a dose of 0.3 mg/kg every alternate day and has since a hemoglobin level of >9.0 g/dL without further RBC transfusions. LESSONS: We learned that a Bochdalek type of CDH can manifest in a DBA patient with RPS19 gene mutation. Therefore, patients diagnosed with the latter disorder should also be screened for an early detection of potential CDHs.


Assuntos
Anemia de Diamond-Blackfan , Células da Medula Óssea/patologia , Transfusão de Eritrócitos/métodos , Hérnias Diafragmáticas Congênitas/diagnóstico , Prednisona/administração & dosagem , Proteínas Ribossômicas/genética , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/fisiopatologia , Exame de Medula Óssea/métodos , Glucocorticoides/administração & dosagem , Humanos , Masculino , Mutação , Radioterapia Assistida por Computador/métodos , Resultado do Tratamento , Adulto Jovem
2.
Nat Commun ; 10(1): 2542, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186416

RESUMO

Somatic ribosomal protein mutations have recently been described in cancer, yet their impact on cellular transcription and translation remains poorly understood. Here, we integrate mRNA sequencing, ribosome footprinting, polysomal RNA sequencing and mass spectrometry datasets from a mouse lymphoid cell model to characterize the T-cell acute lymphoblastic leukemia (T-ALL) associated ribosomal RPL10 R98S mutation. Surprisingly, RPL10 R98S induces changes in protein levels primarily through transcriptional rather than translation efficiency changes. Phosphoserine phosphatase (PSPH), encoding a key serine biosynthesis enzyme, was the only gene with elevated transcription and translation leading to protein overexpression. PSPH upregulation is a general phenomenon in T-ALL patient samples, associated with elevated serine and glycine levels in xenograft mice. Reduction of PSPH expression suppresses proliferation of T-ALL cell lines and their capacity to expand in mice. We identify ribosomal mutation driven induction of serine biosynthesis and provide evidence supporting dependence of T-ALL cells on PSPH.


Assuntos
Glicina/metabolismo , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Serina/metabolismo , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Camundongos , Monoéster Fosfórico Hidrolases , Polirribossomos/genética , Polirribossomos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Análise de Sequência de RNA
3.
Gene ; 706: 69-76, 2019 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-31054365

RESUMO

The receptor for activated c-kinase (RACK1, Asc1 in yeast) is a eukaryotic ribosomal protein located in the head region of the 40S subunit near the mRNA exit channel. This WD-repeat ß-propeller protein acts as a signaling molecule and is involved in metabolic regulation, cell cycle progression, and translational control. However, the exact details of the RACK1 recruitment and stable association with the 40S ribosomal subunit remain only partially known. X-ray analyses of the yeast, Saccharomyces cerevisiae, ribosome revealed that the RACK1 propeller blade (4-5) interacts with the eukaryote-specific C-terminal domain (CTD) of ribosomal protein S3 (uS3 family). To check the functional significance of this interaction, we generated mutant yeast strains harboring C-terminal deletions of uS3. We found that deletion of the 20 C-terminal residues (interacting with blade 4-5) from the uS3-CTD abrogates RACK1 binding to the ribosome. Strains with truncated uS3-CTD exhibited compromised cellular growth and protein synthesis similar to that of RACK1Δ strain, thus suggesting that the uS3-CTD is crucial not only for the recruitment and association of RACK1 with the ribosome, but also for its intracellular function. We suggest that eukaryote-specific RACK1-uS3 interaction has evolved to act as a link between the ribosome and the cellular signaling pathways.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptores de Quinase C Ativada/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ligação ao GTP/genética , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/genética , Receptores de Quinase C Ativada/genética , Proteínas Ribossômicas/genética , Subunidades Ribossômicas Menores de Eucariotos/química , Ribossomos/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
4.
Molecules ; 24(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067825

RESUMO

G-quadruplex (G4) structures are highly stable four-stranded DNA and RNA secondary structures held together by non-canonical guanine base pairs. G4 sequence motifs are enriched at specific sites in eukaryotic genomes, suggesting regulatory functions of G4 structures during different biological processes. Considering the high thermodynamic stability of G4 structures, various proteins are necessary for G4 structure formation and unwinding. In a yeast one-hybrid screen, we identified Slx9 as a novel G4-binding protein. We confirmed that Slx9 binds to G4 DNA structures in vitro. Despite these findings, Slx9 binds only insignificantly to G-rich/G4 regions in Saccharomyces cerevisiae as demonstrated by genome-wide ChIP-seq analysis. However, Slx9 binding to G4s is significantly increased in the absence of Sgs1, a RecQ helicase that regulates G4 structures. Different genetic and molecular analyses allowed us to propose a model in which Slx9 recognizes and protects stabilized G4 structures in vivo.


Assuntos
Proteínas de Ligação a DNA/química , Quadruplex G , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Genoma/genética , Conformação de Ácido Nucleico , Ligação Proteica , RecQ Helicases/química , RecQ Helicases/genética , Proteínas Ribossômicas/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Termodinâmica
5.
mSphere ; 4(2)2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867326

RESUMO

Balancing gene expression is a fundamental challenge of all cell types. To properly regulate transcription on a genome-wide level, there are myriad mechanisms employed by the cell. One layer to this regulation is through spatial positioning, with particular chromosomal loci exerting an influence on transcription throughout a region. Many coregulated gene families utilize spatial positioning to coordinate transcription, with functionally related genes clustering together which can allow coordinated expression via adjacent gene coregulation. The mechanisms underlying this process have not been elucidated, though there are many coregulated gene families that exhibit this genomic distribution. In the present study, we tested for a role for the enhancer-promoter (EP) hypothesis, which demonstrates that regulatory elements can exert transcriptional effects over a broad distance, in coordinating transcriptional coregulation using budding yeast, Saccharomyces cerevisiae We empirically validated the EP model, finding that the genomic distance a promoter can affect varies by locus, which can profoundly affect levels of transcription, phenotype, and the extent of transcriptional disruption throughout a genomic region. Using the nitrogen metabolism, ribosomal protein, toxin response, and heat shock gene families as our test case, we report functionally clustered genes localize to genomic loci that are more conducive to transcriptional regulation at a distance compared to the unpaired members of the same families. Furthermore, we report that the coregulation of functional clusters is dependent, in part, on chromatin maintenance and remodeling, providing one mechanism underlying adjacent gene coregulation.IMPORTANCE The two-dimensional, physical positioning of genes along a chromosome can impact proper transcriptional regulation throughout a genomic region. The transcription of neighboring genes is correlated in a genome-wide manner, which is a characteristic of eukaryotes. Many coregulated gene families can be found clustered with another member of the same set-which can result in adjacent gene coregulation of the pair. Due to the myriad gene families that exhibit a nonrandom genomic distribution, there are likely multiple mechanisms working in concert to properly regulate transcriptional coordination of functionally clustered genes. In this study, we utilized budding yeast in an attempt to elucidate mechanisms that underlie this coregulation: testing and empirically validating the enhancer-promoter hypothesis in this species and reporting that functionally related genes cluster to genomic regions that are more conducive to transcriptional regulation at a distance. These clusters rely, in part, on chromatin maintenance and remodelers to maintain proper transcriptional coordination. Our work provides insight into the mechanisms underlying adjacent gene coregulation.


Assuntos
Genoma Fúngico , Família Multigênica , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Transcrição Genética , Regulação Fúngica da Expressão Gênica , Proteínas de Choque Térmico/genética , Nitrogênio/metabolismo , Proteínas Ribossômicas/genética , Fatores de Transcrição/genética
6.
Theriogenology ; 129: 77-81, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30826720

RESUMO

Ribosomal protein S3 (RpS3), a member of the ribosome 40S subunit, has conventional ribosomal function and additional extraribosomal functions. The aim of the present study was to analyze the expression and localization of RpS3 and its function in early embryogenesis in mice. RpS3 mRNA and protein were expressed in multiple mouse tissues. In the ovary, RpS3 protein was ubiquitously and highly expressed in oocytes and granulosa cells. After ovulation and fertilization, RpS3 mRNA and protein were detected in oocytes and preimplantation embryos. Furthermore, RpS3 protein was localized in the cytoplasm of oocytes and preimplantation embryos. Moreover, knockdown of RpS3 in zygotes led to a significantly decreased rate of blastocyst formation. These results provide the first evidence for a novel function of RpS3 in regulating early embryonic development in mice.


Assuntos
Desenvolvimento Embrionário/genética , Proteínas Ribossômicas/fisiologia , Animais , Blastocisto/metabolismo , Feminino , Técnicas de Silenciamento de Genes/veterinária , Masculino , Camundongos , Camundongos Endogâmicos ICR , Oócitos/metabolismo , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
7.
Chem Biol Interact ; 304: 1-9, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30831090

RESUMO

Ribosomal protein S15A (RPS15A) has emerged as a novel oncogene of various human cancers. However, whether RPS15A is involved in pancreatic cancer remains unclear. In this study, we aimed to investigate the potential relevance of RPS15A in pancreatic cancer and elucidate the underlying regulatory mechanism. We found that RPS15A expression was significantly up-regulated in pancreatic cancer cell lines. RPS15A knockdown resulted in a decrease of cell proliferation and colony formation, and induced cell cycle arrest in G0/G1 phases of pancreatic cancer cells in vitro. In addition, RPS15A knockdown down-regulated ß-catenin expression and blocked the activation of Wnt signaling. Notably, RPS15A was identified as a target gene of microRNA-519d-3p (miR-519d-3p), a tumor suppressive miRNA. Further data showed that miR-519d-3p negatively regulated RPS15A expression in pancreatic cancer cells. Moreover, miR-591d-3p expression was significantly decreased in pancreatic cancer cell lines and tissues and was inversely correlated with RPS15A expression. The overexpression of miR-519d-3p significantly inhibited the proliferation and Wnt/ß-catenin signaling in pancreatic cancer cells, mimicking the similar effect of RPS15A knockdown. However, restoration of RPS15A expression partially reversed the antitumor effect of miR-519d-3p. Taken together, our results demonstrate that RPS15A knockdown or RPS15A inhibition by miR-519d-3p suppresses the growth of pancreatic cancer cells associated with the inhibition of Wnt/ß-catenin signaling. Our study suggests that the miR-519d-3p/RPS15A/Wnt/ß-catenin regulation axis plays an important role in the progression of pancreatic cancer and may serve as potential targets for treatment of pancreatic cancer.


Assuntos
MicroRNAs/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Ribossômicas/genética , Via de Sinalização Wnt/genética , Proliferação de Células/efeitos dos fármacos , Células HEK293 , Humanos , MicroRNAs/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Ribossômicas/metabolismo , Células Tumorais Cultivadas
8.
Theriogenology ; 130: 120-124, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30884332

RESUMO

Microminipigs are one of the smallest miniature pigs characterized as sexually precocious; the males achieve sexual maturity at around 3-4.5 months of age. However, the physiology of this sexual precocity is still unclear. To understand sexual precocity in male microminipigs, we analyzed their testes at five developmental stages: neonatal (<7 days), 30-day-old, 45-day-old, 80-day-old, and adult (>24 months) stages. We used 4 pigs in each of the stages. To analyze testicular development histologically, the seminiferous tubule diameter (SD) was measured, and the presence or absence of the seminiferous lumen was confirmed. Changes in the expression of pluripotency markers, DBA, UCHL1, ZBTB16, and vimentin, were evaluated immunohistologically. For the analyses, cells positive for DBA, UCHL1, and ZBTB16 per 150 round seminiferous tubules in cross sections from each testis were counted to evaluate the total number of positive cells. The number of positive cells per 100 Sertoli cells (DBA+/Sertoli, UCHL1+/Sertoli, and ZBTB16+/Sertoli) was calculated to compare the five developmental stages. Histologically, SDs became larger with piglet growth, and precocity was confirmed; seminiferous lumens were observed from the 30-day-old stage. Immunohistologically, the number of DBA+/Sertoli, which indicates the number of gonocytes, decreased rapidly to an undetectable level by the 45-day-old stage. In the same period, the number of UCHL1+/Sertoli, which indicates total SSCs, increased significantly, suggesting that the proliferation of SSCs was accelerated before 30 days of age. Consequently, our study clarified that differentiation of SSCs in microminipigs started during the fetal period, the differentiation of gonocytes and proliferation of SSCs was then accelerated before 30 days of age, and the early phase of spermatogenesis was finally completed at around 45 days after birth. Consequently, sexual precocity in male microminipigs was characterized by a shorter duration of the early phase of spermatogenesis.


Assuntos
Células-Tronco Germinativas Adultas/metabolismo , Maturidade Sexual/fisiologia , Suínos/fisiologia , Animais , Biomarcadores , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Espermatogênese/fisiologia , Porco Miniatura , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Vimentina/genética , Vimentina/metabolismo
9.
Mitochondrial DNA A DNA Mapp Seq Anal ; 30(3): 573-584, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30845854

RESUMO

In the mitochondrial genomes of the filamentous Ascomycota, aside from the usual 'core' set of genes, one can encounter genes encoding for ribosomal protein S3 (rps3), N-acetyltransferase, and in a few instances aminotransferases. Based on a survey using sequence data from various databases, it was observed that these genes can be located within introns or exist as freestanding genes in intergenic regions. Furthermore, they can also be absent from fungal mitochondrial genomes. The rps3 gene is highly conserved among fungal mitochondrial genomes although examples were noted where the mtDNA version of this gene has been translocated into the nuclear genome. The N-acetyltransferase gene was less frequently encountered and may be a more recent import from the nuclear genome. Both genes serve as examples of genetic elements that appear to be capable of 'cycling' or mobilizing between introns and intergenic regions and possible between the nuclear and mitochondrial genomes. This 'cycling' mechanism is currently not understood but may involve recombination events and/or movement via RNA intermediates.


Assuntos
Acetiltransferases/genética , Ascomicetos/genética , Genes Mitocondriais/genética , Íntrons/genética , Proteínas Ribossômicas/genética , Acetiltransferases/metabolismo , Ascomicetos/metabolismo
10.
Int J Oncol ; 54(5): 1591-1600, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30816492

RESUMO

Drug resistance is a major cause of cancer­associated mortality. Epirubicin­based chemotherapy initially benefits patients with metastatic or advanced gastric cancer; however, tumor recurrence can occur following several courses of treatment. Mitochondrial ribosomal protein L33 (MRPL33)­long (L) and MRPL33­short (S), isoforms of MRPL33 that arise from AS, have been reported to regulate cell growth and apoptosis in cancer; however, few studies have evaluated the roles of MRPL33­L and MRPL33­S in gastric cancer. In the present study, MRPL33­L was demonstrated to be significantly more abundant in gastric tumor tissues than the MRPL33­S isoform. MRPL33­S promoted chemosensitivity to epirubicin in gastric cancer as demonstrated by a chemoresponse assay; chemosensitivity was suppressed in response to MRPL33­L. Gene microarray analysis was performed to investigate the underlying mechanisms. Bioinformatic analysis revealed that overexpression of MRPL33­L and MRPL33­S served critical roles in transcription, signal transduction and apoptosis. In particular, the phosphoinositide 3­kinase (PI3K)/AKT serine/threonine kinase (AKT) signaling pathway was markedly regulated. A total of 36 target genes, including PIK3 regulatory subunit α, AKT2, cAMP response element­binding protein (CREB) 1, forkhead box 3, glycogen synthase kinase 3ß and mammalian target of rapamycin, which are involved in the PI3K/AKT signaling pathway, were selected for further investigation via protein­protein interaction network and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Furthermore, western blot analysis indicated that MRPL33­S promoted the chemoresponse to epirubicin by deactivating PI3K/AKT/CREB signaling and inducing apoptosis, while MRPL33­L had the opposite effects. In conclusion, the results of the present study revealed that isoforms S and L of MRPL33, which arise from alternative splicing, exhibited opposing roles in the chemoresponse to epirubicin in gastric cancer via the PI3K/AKT signaling pathway. These findings may contribute to the development of potential therapeutic strategies for the resensitization of patients with gastric cancer to epirubicin treatment.


Assuntos
Processamento Alternativo , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica/métodos , Proteínas Mitocondriais/genética , Proteínas Ribossômicas/genética , Neoplasias Gástricas/genética , Regulação para Cima , Adulto , Idoso , Linhagem Celular Tumoral , Epirubicina/uso terapêutico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo
11.
Plant Sci ; 281: 93-101, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30824066

RESUMO

The extraordinary incidence of Horizontal Gene Transfer (HGT) mostly in mitochondrial genomes of flowering plants is well known. Here, we report another episode of HGT affecting a large mitochondrial gene region in the evergreen conifer Atlas cedar (Cedrus atlantica). Mitochondria of this Pinaceae species possess an rps3 gene that harbours two introns and shares the same genomic context with a downstream overlapping rpl16 gene, like in the major groups of gymnosperms and angiosperms analyzed so far. Interestingly, C. atlantica contains additional copies of the rps3 and rpl16 sequences that are more closely related to angiosperm counterparts than to those from gymnosperms, as also confirmed by phylogenetic analyses. This suggests that a lateral transfer from a flowering plant donor is the most likely mechanism for the origin of the Atlas cedar extra sequences. Quantitative PCR and reverse-transcription (RT)-PCR analyses demonstrate, respectively, mitochondrial location and lack of expression for the rps3 and rpl16 additional sequences in C. atlantica. Furthermore, our study provides evidence that a similar HGT event takes place in two other Cedrus species, which occurr in Cyprus and North Africa. Only the West Himalayan C. deodara lacks the transferred genes. The potential donor and the molecular mechanism underlying this lateral DNA transfer remain still unclear.


Assuntos
Cedrus/genética , Transferência Genética Horizontal/genética , Genoma Mitocondrial/genética , Proteínas Ribossômicas/genética
12.
Mol Biol Evol ; 36(5): 1071-1085, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30835268

RESUMO

Repeated evolution of functionally similar phenotypes is observed throughout the tree of life. The extent to which the underlying genetics are conserved remains an area of considerable interest. Previously, we reported the evolution of colony switching in two independent lineages of Pseudomonas fluorescens SBW25. The phenotypic and genotypic bases of colony switching in the first lineage (Line 1) have been described elsewhere. Here, we deconstruct the evolution of colony switching in the second lineage (Line 6). We show that, as for Line 1, Line 6 colony switching results from an increase in the expression of a colanic acid-like polymer (CAP). At the genetic level, nine mutations occur in Line 6. Only one of these-a nonsynonymous point mutation in the housekeeping sigma factor rpoD-is required for colony switching. In contrast, the genetic basis of colony switching in Line 1 is a mutation in the metabolic gene carB. A molecular model has recently been proposed whereby the carB mutation increases capsulation by redressing the intracellular balance of positive (ribosomes) and negative (RsmAE/CsrA) regulators of a positive feedback loop in capsule expression. We show that Line 6 colony switching is consistent with this model; the rpoD mutation generates an increase in ribosomal gene expression, and ultimately an increase in CAP expression.


Assuntos
Evolução Biológica , Fenótipo , Pseudomonas fluorescens/genética , Cápsulas Bacterianas/fisiologia , Epistasia Genética , Regulação Bacteriana da Expressão Gênica , Mutação , Proteínas Ribossômicas/genética
13.
Biol Res ; 52(1): 4, 2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30717818

RESUMO

BACKGROUND: Hematoporphyrin derivative (HPD) has a sensibilization effect in lung adenocarcinoma. This study was conducted to identify the target genes of HPD in lung adenocarcinoma. METHODS: RNA sequencing was performed using the lung adenocarcinoma cell line A549 after no treatment or treatment with X-ray or X-ray + HPD. The differentially expressed genes (DEGs) were screened using Mfuzz package by noise-robust soft clustering analysis. Enrichment analysis was carried out using "BioCloud" online tool. Protein-protein interaction (PPI) network and module analyses were performed using Cytoscape software. Using WebGestalt tool and integrated transcription factor platform (ITFP), microRNA target and transcription factor (TF) target pairs were separately predicted. An integrated regulatory network was visualized with Cytoscape software. RESULTS: A total of 815 DEGs in the gene set G1 (continuously dysregulated genes along with changes in processing conditions [untreated-treated with X-ray-X-ray + treated with HPD]) and 464 DEGs in the gene set G2 (significantly dysregulated between X-ray + HPD-treated group and untreated/X-ray-treated group) were screened. The significant module identified from the PPI network for gene set G1 showed that ribosomal protein L3 (RPL3) gene could interact with heat shock protein 90 kDa alpha, class A member 1 (HSP90AA1). TFs AAA domain containing 2 (ATAD2) and protein inhibitor of activated STAT 1 (PIAS1) were separately predicted for the genes in gene set G1 and G2, respectively. In the integrated network for gene set G2, ubiquitin-specific peptidase 25 (USP25) was targeted by miR-200b, miR-200c, and miR-429. CONCLUSION: RPL3, HSP90AA1, ATAD2, and PIAS1 as well as USP25, which is targeted by miR-200b, miR-200c, and miR-429, may be the potential targets of HPD in lung adenocarcinoma.


Assuntos
Adenocarcinoma de Pulmão/genética , Redes Reguladoras de Genes/genética , Derivado da Hematoporfirina/farmacologia , Neoplasias Pulmonares/genética , ATPases Associadas a Diversas Atividades Celulares/efeitos dos fármacos , ATPases Associadas a Diversas Atividades Celulares/genética , Adenocarcinoma de Pulmão/tratamento farmacológico , Adenocarcinoma de Pulmão/radioterapia , Linhagem Celular Tumoral , Análise por Conglomerados , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP90/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapia , MicroRNAs/metabolismo , Proteínas Inibidoras de STAT Ativados/efeitos dos fármacos , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Ribossômicas/efeitos dos fármacos , Proteínas Ribossômicas/genética , Análise de Sequência de RNA , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/efeitos dos fármacos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Fatores de Transcrição
14.
RNA ; 25(5): 521-538, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30733326

RESUMO

It has recently become clear that ribosomes are much more heterogeneous than previously thought, with diversity arising from rRNA sequence and modifications, ribosomal protein (RP) content and posttranslational modifications (PTMs), as well as bound nonribosomal proteins. In some cases, the existence of these diverse ribosome populations has been verified by biochemical or structural methods. Furthermore, knockout or knockdown of RPs can diversify ribosome populations, while also affecting the translation of some mRNAs (but not others) with biological consequences. However, the effects on translation arising from depletion of diverse proteins can be highly similar, suggesting that there may be a more general defect in ribosome function or stability, perhaps arising from reduced ribosome numbers. Consistently, overall reduced ribosome numbers can differentially affect subclasses of mRNAs, necessitating controls for specificity. Moreover, in order to study the functional consequences of ribosome diversity, perturbations including affinity tags and knockouts are introduced, which can also affect the outcome of the experiment. Here we review the available literature to carefully evaluate whether the published data support functional diversification, defined as diverse ribosome populations differentially affecting translation of distinct mRNA (classes). Based on these observations and the commonly observed cellular responses to perturbations in the system, we suggest a set of important controls to validate functional diversity, which should include gain-of-function assays and the demonstration of inducibility under physiological conditions.


Assuntos
Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA de Transferência/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Animais , Archaea/genética , Archaea/metabolismo , Bactérias/genética , Bactérias/metabolismo , Sequência de Bases , Heterogeneidade Genética , Mamíferos/genética , Mamíferos/metabolismo , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Proteínas Ribossômicas/genética , Ribossomos/classificação , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
15.
RNA ; 25(4): 472-480, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30705137

RESUMO

In vitro reconstitution studies have shown that ribosome assembly is highly cooperative and starts with the binding of a few ribosomal (r-) proteins to rRNA. It is unknown how these early binders act. Focusing on the initial stage of the assembly of the large subunit of the Escherichia coli ribosome, we prepared a 79-nucleotide-long region of 23S rRNA encompassing the binding sites of the early binders uL4 and uL24. Force signals were measured in a DNA/RNA dumbbell configuration with a double optical tweezers setup. The rRNA fragment was stretched until unfolded, in the absence or in the presence of the r-proteins (either uL4, uL24, or both). We show that the r-proteins uL4 and uL24 individually stabilize the rRNA fragment, both acting as molecular clamps. Interestingly, this mechanical stabilization is enhanced when both proteins are bound simultaneously. Independently, we observe a cooperative binding of uL4 and uL24 to the rRNA fragment. These two aspects of r-proteins binding both contribute to the efficient stabilization of the 3D structure of the rRNA fragment under investigation. We finally consider implications of our results for large ribosomal subunit assembly.


Assuntos
RNA Bacteriano/química , RNA Ribossômico 23S/química , Proteínas Ribossômicas/genética , Ribossomos/química , Pareamento de Bases , Sequência de Bases , Fenômenos Biomecânicos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Pinças Ópticas , Biogênese de Organelas , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
17.
Dev Cell ; 48(6): 811-826.e6, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30799226

RESUMO

Caenorhabditis elegans provides an amenable system to explore whether newly composed ribosomes are required to progress through development. Despite the complex pattern of tissues that are formed during embryonic development, we found that null homozygotes lacking any of the five different ribosomal proteins (RPs) can produce fully functional first-stage larvae, with similar developmental competence seen upon complete deletion of the multi-copy ribosomal RNA locus. These animals, relying on maternal but not zygotic contribution of ribosomal components, are capable of completing embryogenesis. In the absence of new ribosomal components, the resulting animals are arrested before progression from the first larval stage and fail in two assays for postembryonic plasticity of neuronal structure. Mosaic analyses of larvae that are a mixture of ribosome-competent and non-competent cells suggest a global regulatory mechanism in which ribosomal insufficiency in a subset of cells triggers organism-wide growth arrest.


Assuntos
Caenorhabditis elegans/embriologia , Desenvolvimento Embrionário , Organogênese , Ribossomos/metabolismo , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Larva/metabolismo , Mosaicismo , Mutação/genética , Fenótipo , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Transcrição Genética , Zigoto/metabolismo
18.
Nat Commun ; 10(1): 930, 2019 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-30804338

RESUMO

Ribo-T is an engineered ribosome whose small and large subunits are tethered together by linking 16S rRNA and 23S rRNA in a single molecule. Although Ribo-T can support cell proliferation in the absence of wild type ribosomes, Ribo-T cells grow slower than those with wild type ribosomes. Here, we show that cell growth defect is likely explained primarily by slow Ribo-T assembly rather than its imperfect functionality. Ribo-T maturation is stalled at a late assembly stage. Several post-transcriptional rRNA modifications and some ribosomal proteins are underrepresented in the accumulated assembly intermediates and rRNA ends are incompletely trimmed. Ribosome profiling of Ribo-T cells shows no defects in translation elongation but reveals somewhat higher occupancy by Ribo-T of the start codons and to a lesser extent stop codons, suggesting that subunit tethering mildly affects the initiation and termination stages of translation. Understanding limitations of Ribo-T system offers ways for its future development.


Assuntos
Subunidades Ribossômicas/química , Subunidades Ribossômicas/metabolismo , Códon de Iniciação/genética , Códon de Iniciação/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genética , RNA Ribossômico 23S/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas/genética
19.
Nat Rev Cancer ; 19(4): 228-238, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30670820

RESUMO

Long thought to be too big and too ubiquitous to fail, we now know that human cells can fail to make sufficient amounts of ribosomes, causing a number of diseases collectively known as ribosomopathies. The best characterized ribosomopathies, with the exception of Treacher Collins syndrome, are inherited bone marrow failure syndromes, each of which has a marked increase in cancer predisposition relative to the general population. Although rare, emerging data reveal that the inherited bone marrow failure syndromes may be underdiagnosed on the basis of classical symptomology, leaving undiagnosed patients with these syndromes at an elevated risk of cancer without adequate counselling and surveillance. The link between the inherited ribosomopathies and cancer has led to greater awareness that somatic mutations in factors involved in ribosome biogenesis may also be drivers in sporadic cancers. Our goal here is to compare and contrast the pathophysiological mechanisms underpinning ribosomopathies to gain a better understanding of the mechanisms that predispose these disorders to cancer.


Assuntos
Neoplasias/genética , Neoplasias/patologia , Ribossomos/genética , Ribossomos/patologia , Animais , Predisposição Genética para Doença/genética , Humanos , Mutação/genética , Proteínas Ribossômicas/genética
20.
J Biol Chem ; 294(8): 2827-2838, 2019 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-30598506

RESUMO

Ribosomal proteins are the building blocks of ribosome biogenesis. Beyond their known participation in ribosome assembly, the ribosome-independent functions of ribosomal proteins are largely unknown. Here, using immunoprecipitation, subcellular fractionation, His-ubiquitin pulldown, and immunofluorescence microscopy assays, along with siRNA-based knockdown approaches, we demonstrate that ribosomal protein L6 (RPL6) directly interacts with histone H2A and is involved in the DNA damage response (DDR). We found that in response to DNA damage, RPL6 is recruited to DNA damage sites in a poly(ADP-ribose) polymerase (PARP)-dependent manner, promoting its interaction with H2A. We also observed that RPL6 depletion attenuates the interaction between mediator of DNA damage checkpoint 1 (MDC1) and H2A histone family member X, phosphorylated (γH2AX), impairs the accumulation of MDC1 at DNA damage sites, and reduces both the recruitment of ring finger protein 168 (RNF168) and H2A Lys-15 ubiquitination (H2AK15ub). These RPL6 depletion-induced events subsequently inhibited the recruitment of the following downstream repair proteins: tumor protein P53-binding protein 1 (TP53BP1) and BRCA1, DNA repair-associated (BRCA1). Moreover, the RPL6 knockdown resulted in defects in the DNA damage-induced G2-M checkpoint, DNA damage repair, and cell survival. In conclusion, our study identifies RPL6 as a critical regulatory factor involved in the DDR. These findings expand our knowledge of the extraribosomal functions of ribosomal proteins in cell physiology and deepen our understanding of the molecular mechanisms underlying DDR regulation.


Assuntos
Proteína BRCA1/metabolismo , Dano ao DNA , Reparo do DNA , Histonas/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas Ribossômicas/metabolismo , Proteína BRCA1/genética , Ciclo Celular , Sobrevivência Celular , Células HEK293 , Células HeLa , Histonas/genética , Humanos , Poli(ADP-Ribose) Polimerase-1/genética , Proteínas Ribossômicas/genética , Transdução de Sinais , Ubiquitina , Ubiquitinação
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