RESUMO
Transcription factors play key roles in development and disease by controlling gene expression. Forkhead box A1 (FOXA1), is a pioneer transcription factor essential for mouse development and functions as an oncogene in prostate and breast cancer. In colorectal cancer (CRC), FOXA1 is significantly downregulated and high FOXA1 expression is associated with better prognosis, suggesting potential tumor suppressive functions. We therefore investigated the regulation of FOXA1 expression in CRC, focusing on well-differentiated CRC cells, where FOXA1 is robustly expressed. Genome-wide RNA stability assays identified FOXA1 as an unstable mRNA in CRC cells. We validated FOXA1 mRNA instability in multiple CRC cell lines and in patient-derived CRC organoids, and found that the FOXA1 3'UTR confers instability to the FOXA1 transcript. RNA pulldowns and mass spectrometry identified Staufen1 (STAU1) as a potential regulator of FOXA1 mRNA. Indeed, STAU1 knockdown resulted in increased FOXA1 mRNA and protein expression due to increased FOXA1 mRNA stability. Consistent with these data, RNA-seq following STAU1 knockdown in CRC cells revealed that FOXA1 targets were upregulated upon STAU1 knockdown. Collectively, this study uncovers a molecular mechanism by which FOXA1 is regulated in CRC cells and provides insights into our understanding of the complex mechanisms of gene regulation in cancer.
Assuntos
Neoplasias Colorretais , Transcriptoma , Masculino , Humanos , Animais , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Neoplasias Colorretais/metabolismo , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Sigma-1 receptor (S1R) is a calcium-sensitive, ligand-operated receptor chaperone present on the endoplasmic reticulum (ER) membrane. S1R plays an important role in ER-mitochondrial inter-organelle calcium signaling and cell survival. S1R and its agonists confer resilience against various neurodegenerative diseases; however, the molecular mechanism of S1R is not yet fully understood. At resting state, S1R is either in a monomeric or oligomeric state but the ratio of these concentrations seems to change upon activation of S1R. S1R is activated by either cellular stress, such as ER-calcium depletion, or ligands. While the effect of ligands on S1R quaternary structure remains unclear, the effect of cellular stress has not been studied. In this study we utilize cellular and an in-vivo model to study changes in quaternary structure of S1R upon activation. We incubated cells with cellular stressors (H2O2 and thapsigargin) or exogenous ligands, then quantified monomeric and oligomeric forms. We observed that benzomorphan-based S1R agonists induce monomerization of S1R and decrease oligomerization, which was confirmed in the liver tissue of mice injected with (+)-Pentazocine. Antagonists block this effect but do not induce any changes when used alone. Oxidative stress (H2O2) increases the monomeric/oligomeric S1R ratio whereas ER calcium depletion (thapsigargin) has no effect. We also analyzed the oligomerization ability of various truncated S1R fragments and identified the fragments favorizing oligomerization. In this publication we demonstrate that quaternary structural changes differ according to the mechanism of S1R activation. Therefore, we offer a novel perspective on S1R activation as a nuanced phenomenon dependent on the type of stimulus.
Assuntos
Benzomorfanos , Cálcio , Animais , Camundongos , Peróxido de Hidrogênio , Receptor Sigma-1 , Tapsigargina , Sinalização do CálcioRESUMO
Matrin3 is an RNA-binding protein that regulates diverse RNA-related processes, including mRNA splicing. Although Matrin3 has been intensively studied in neurodegenerative diseases, its function in cancer remains unclear. Here, we report Matrin3-mediated regulation of mitotic spindle dynamics in colorectal cancer (CRC) cells. We comprehensively identified RNAs bound and regulated by Matrin3 in CRC cells and focused on CDC14B, one of the top Matrin3 targets. Matrin3 knockdown results in increased inclusion of an exon containing a premature termination codon in the CDC14B transcript and simultaneous down-regulation of the standard CDC14B transcript. Knockdown of CDC14B phenocopies the defects in mitotic spindle dynamics upon Matrin3 knockdown, and the elongated and misoriented mitotic spindle observed upon Matrin3 knockdown are rescued upon overexpression of CDC14B, suggesting that CDC14B is a key downstream effector of Matrin3. Collectively, these data reveal a role for the Matrin3/CDC14B axis in control of mitotic spindle dynamics.
Assuntos
Processamento Alternativo , Fosfatases de Especificidade Dupla , Processamento Alternativo/genética , Fosfatases de Especificidade Dupla/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fuso Acromático/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMO
PURPL is a p53-induced lncRNA that suppresses basal p53 levels. Here, we investigated PURPL upon p53 activation in liver cancer cells, where it is expressed at significantly higher levels than other cell types. Using isoform sequencing, we discovered novel PURPL transcripts that have a retained intron and/or previously unannotated exons. To determine PURPL function upon p53 activation, we performed transcriptome sequencing (RNA-Seq) after depleting PURPL using CRISPR interference (CRISPRi), followed by Nutlin treatment to induce p53. Strikingly, although loss of PURPL in untreated cells altered the expression of only 7 genes, loss of PURPL resulted in altered expression of ~800 genes upon p53 activation, revealing a context-dependent function of PURPL. Pathway analysis suggested that PURPL is important for fine-tuning the expression of specific genes required for mitosis. Consistent with these results, we observed a significant decrease in the percentage of mitotic cells upon PURPL depletion. Collectively, these data identify novel transcripts from the PURPL locus and suggest that PURPL delicately moderates the expression of mitotic genes in the context of p53 activation to control cell cycle arrest.
Assuntos
RNA Longo não Codificante , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transcriptoma/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Éxons/genéticaRESUMO
DNA helicase RECQ1 (also known as RECQL or RECQL1) is a candidate breast cancer susceptibility gene significantly correlated with clinical outcomes of sporadic breast cancer patients. Prior studies have suggested that RECQ1 maintains genomic stability by regulating a wide variety of core cellular functions including DNA replication, DNA damage response, and transcription. However, it is unclear which, if any, of these are the primary functions of RECQ1 as related to its role in suppressing breast cancer. We describe here an unbiased integrative genomics approach that enabled us to discover a previously unknown regulatory role of RECQ1 in promoting Estrogen Receptor alpha (ERα) expression and the expression of specific ERα target genes in ER positive breast cancer cells. We discuss potential future applications of similar experimental strategies in advancing the mechanistic understanding and elucidating specific new details of genome-wide functions of RECQ1 and other RecQ helicases in maintaining genomic stability and preventing cancer.
Assuntos
Neoplasias da Mama , RecQ Helicases , Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Feminino , Predisposição Genética para Doença , Instabilidade Genômica , Humanos , RecQ Helicases/genéticaRESUMO
One of the primary mechanisms of post-transcriptional gene regulation is the modulation of RNA stability. We recently discovered that LINC00675, a transcript annotated as a long noncoding RNA (lncRNA), is transcriptionally regulated by FOXA1 and encodes a highly conserved small protein that localizes to the endoplasmic reticulum, hence renamed as FORCP (FOXA1-regulated conserved small protein). Here, we show that the endogenous FORCP transcript is rapidly degraded and rendered unstable as a result of 3'UTR-mediated degradation. Surprisingly, although the FORCP transcript is a canonical nonsense-mediated decay (NMD) and microRNA (miRNA) target, we found that it is not degraded by NMD or miRNAs. Targeted deletion of an evolutionarily conserved region in the FORCP 3'UTR using CRISPR/Cas9 significantly increased the stability of the FORCP transcript. Interestingly, this region requires the presence of an immediate downstream 55-nt-long sequence for transcript stability regulation. Functionally, colorectal cancer cells lacking this conserved region expressed from the endogenous FORCP locus displayed decreased proliferation and clonogenicity. These data demonstrate that the FORCP transcript is destabilized via conserved elements within its 3'UTR and emphasize the need to interrogate the function of a given 3'UTR in its native context.
Assuntos
RNA Longo não Codificante , Regiões 3' não Traduzidas/genética , Elementos Ricos em Adenilato e Uridilato , Regulação da Expressão Gênica , Estabilidade de RNA/genética , RNA Longo não Codificante/genéticaRESUMO
p53, master transcriptional regulator of the genotoxic stress response, controls cell-cycle arrest and apoptosis following DNA damage. Here, we identify a p53-induced lncRNA suicidal PARP-1 cleavage enhancer (SPARCLE) adjacent to miR-34b/c required for p53-mediated apoptosis. SPARCLE is a â¼770-nt, nuclear lncRNA induced 1 day after DNA damage. Despite low expression (<16 copies/cell), SPARCLE deletion increases DNA repair and reduces DNA-damage-induced apoptosis as much as p53 deficiency, while its overexpression restores apoptosis in p53-deficient cells. SPARCLE does not alter gene expression. SPARCLE binds to PARP-1 with nanomolar affinity and causes apoptosis by acting as a caspase-3 cofactor for PARP-1 cleavage, which separates PARP-1's N-terminal (NT) DNA-binding domain from its catalytic domains. NT-PARP-1 inhibits DNA repair. Expressing NT-PARP-1 in SPARCLE-deficient cells increases unrepaired DNA damage and restores apoptosis after DNA damage. Thus, SPARCLE enhances p53-induced apoptosis by promoting PARP-1 cleavage, which interferes with DNA-damage repair.
Assuntos
Apoptose , Caspase 3/metabolismo , Neoplasias Colorretais/enzimologia , Quebras de DNA de Cadeia Dupla , Quebras de DNA de Cadeia Simples , Poli(ADP-Ribose) Polimerase-1/metabolismo , RNA Longo não Codificante/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células A549 , Animais , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HEK293 , Células Hep G2 , Humanos , Masculino , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , RNA Longo não Codificante/genética , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
Long noncoding RNAs (lncRNAs) have emerged as regulators of diverse cellular processes. Although the vast majority of lncRNAs are expressed at lower levels compared to messenger RNAs (mRNAs), many lncRNAs play a central role in the regulation of cellular homeostasis and gene expression. With the advancement of next generation sequencing technologies, recent studies illustrate the diversity of lncRNA function. This diversity can be due to differences in their mechanisms of action, spatio-temporal expression, and/or abundance, all of which can vary depending on the particular cell type or tissue. Here, we discuss how the abundance of lncRNAs is an important feature that is often linked to their functions, and why it is crucial to quantitate lncRNA abundance, its local concentration within a cell or a tissue or the dynamic changes in expression levels during cell cycle progression or upon environmental stimuli, to shed light on their physiological roles.
Assuntos
RNA Longo não Codificante , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genéticaRESUMO
In recent years, long noncoding RNAs (lncRNAs) have been increasingly recognized as critical regulators of a broad spectrum of cellular processes. Recent advancements in proteomic technologies have uncovered that an abundance of noncoding genes, including lncRNAs, have been misannotated and in reality encode proteins. This revelation underscores the need to accurately determine the coding potential of lncRNAs prior to assessment of their functional mechanisms. Here, we detail numerous experimental techniques useful in the determination of lncRNA coding potential. Several of these methods are doubly useful in that they may also be employed in studying the function of a lncRNA, be it via an RNA, protein, or both.
Assuntos
Regulação da Expressão Gênica , Biossíntese de Proteínas , RNA Longo não Codificante/genética , Animais , Sistemas CRISPR-Cas , Biologia Computacional/métodos , Humanos , Anotação de Sequência Molecular , Fases de Leitura Aberta , Polirribossomos/metabolismoRESUMO
Long noncoding RNAs (lncRNAs) are an increasing focus of investigation due to their implications in diverse biological processes and disease. Nevertheless, the majority of lncRNAs are low in abundance and poorly conserved, posing challenges to functional studies. The CRISPR/Cas system, an innovative technology that has emerged over the last decade, can be utilized to further understand lncRNA function. The system targets specific DNA and/or RNA sequences via a guide RNA (gRNA) and Cas nuclease complex. We and others have utilized this technology in various applications such as lncRNA knockout, knockdown, overexpression, and imaging. In this review, we summarize how the CRISPR/Cas technology provides new tools to investigate the roles and therapeutic implications of lncRNAs.
Assuntos
Técnicas de Silenciamento de Genes/métodos , RNA Longo não Codificante/genética , Animais , Sistemas CRISPR-Cas , HumanosRESUMO
Mammalian circRNAs can influence different cellular processes by interacting with proteins and other nucleic acids. Here, we used ribonucleoprotein immunoprecipitation (RIP) analysis to identify systematically the circRNAs associated with the cancer-related protein AUF1. Among the circRNAs interacting with AUF1 in HeLa (human cervical carcinoma) cells, we focused on hsa_circ_0032434 (circPCNX), an abundant target of AUF1. Overexpression of circPCNX specifically interfered with the binding of AUF1 to p21 (CDKN1A) mRNA, thereby promoting p21 mRNA stability and elevating the production of p21, a major inhibitor of cell proliferation. Conversely, silencing circPCNX increased AUF1 binding to p21 mRNA, reducing p21 production and promoting cell division. Importantly, eliminating the AUF1-binding region of circPCNX abrogated the rise in p21 levels and rescued proliferation. Therefore, we propose that the interaction of circPCNX with AUF1 selectively prevents AUF1 binding to p21 mRNA, leading to enhanced p21 mRNA stability and p21 protein production, thereby suppressing cell growth.
Assuntos
Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , RNA Circular/metabolismo , Regiões 3' não Traduzidas , Sítios de Ligação , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células HeLa , Humanos , RNA Circular/química , RNA Mensageiro/metabolismoRESUMO
Susceptibility to breast cancer is significantly increased in individuals with germ line mutations in RECQ1 (also known as RECQL or RECQL1), a gene encoding a DNA helicase essential for genome maintenance. We previously reported that RECQ1 expression predicts clinical outcomes for sporadic breast cancer patients stratified by estrogen receptor (ER) status. Here, we utilized an unbiased integrative genomics approach to delineate a cross talk between RECQ1 and ERα, a known master regulatory transcription factor in breast cancer. We found that expression of ESR1, the gene encoding ERα, is directly activated by RECQ1. More than 35% of RECQ1 binding sites were cobound by ERα genome-wide. Mechanistically, RECQ1 cooperates with FOXA1, the pioneer transcription factor for ERα, to enhance chromatin accessibility at the ESR1 regulatory regions in a helicase activity-dependent manner. In clinical ERα-positive breast cancers treated with endocrine therapy, high RECQ1 and high FOXA1 coexpressing tumors were associated with better survival. Collectively, these results identify RECQ1 as a novel cofactor for ERα and uncover a previously unknown mechanism by which RECQ1 regulates disease-driving gene expression in ER-positive breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Estrogênios/metabolismo , RecQ Helicases/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Predisposição Genética para Doença/genética , Fator 3-alfa Nuclear de Hepatócito/genética , HumanosRESUMO
p53 is an intensely studied tumor-suppressive transcription factor. Recent studies suggest that the RNA-binding protein (RBP) ZMAT3 is important in mediating the tumor-suppressive effects of p53. Here, we globally identify ZMAT3-regulated RNAs and their binding sites at nucleotide resolution in intact colorectal cancer (CRC) cells. ZMAT3 binds to thousands of mRNA precursors, mainly at intronic uridine-rich sequences and affects their splicing. The strongest alternatively spliced ZMAT3 target was CD44, a cell adhesion gene and stem cell marker that controls tumorigenesis. Silencing ZMAT3 increased inclusion of CD44 variant exons, resulting in significant up-regulation of oncogenic CD44 isoforms (CD44v) and increased CRC cell growth that was rescued by concurrent knockdown of CD44v Silencing p53 phenocopied the loss of ZMAT3 with respect to CD44 alternative splicing, suggesting that ZMAT3-mediated regulation of CD44 splicing is vital for p53 function. Collectively, our findings uncover a p53-ZMAT3-CD44 axis in growth suppression in CRC cells.
Assuntos
Processamento Alternativo/genética , Receptores de Hialuronatos/genética , Splicing de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Carcinogênese/genética , Neoplasias Colorretais/genética , Técnicas de Silenciamento de Genes , Inativação Gênica , Células HCT116 , Células HEK293 , Humanos , Receptores de Hialuronatos/metabolismo , Ligação Proteica/genética , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Long noncoding RNAs (lncRNAs) are often associated with polysomes, indicating coding potential. However, only a handful of endogenous proteins encoded by putative lncRNAs have been identified and assigned a function. Here, we report the discovery of a putative gastrointestinal-tract-specific lncRNA (LINC00675) that is regulated by the pioneer transcription factor FOXA1 and encodes a conserved small protein of 79 amino acids which we termed FORCP (FOXA1-Regulated Conserved Small Protein). FORCP transcript is undetectable in most cell types but is abundant in well-differentiated colorectal cancer (CRC) cells where it functions to inhibit proliferation, clonogenicity, and tumorigenesis. The epitope-tagged and endogenous FORCP protein predominantly localizes to the endoplasmic reticulum (ER). In response to ER stress, FORCP depletion results in decreased apoptosis. Our findings on the initial characterization of FORCP demonstrate that FORCP is a novel, conserved small protein encoded by a mis-annotated lncRNA that regulates apoptosis and tumorigenicity in well-differentiated CRC cells.
Assuntos
Apoptose/genética , Carcinogênese/genética , Neoplasias Colorretais/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , RNA Longo não Codificante/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/patologia , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Trato Gastrointestinal/metabolismo , Genes Reporter , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Anotação de Sequência Molecular , Especificidade de Órgãos , RNA Longo não Codificante/genéticaRESUMO
Cell cycle is a cellular process that is subject to stringent control. In contrast to the wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA, SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1 facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle progression via modulating the expression of genes controlling cell proliferation.
Assuntos
Proliferação de Células/genética , Proteínas Correpressoras/genética , Proteínas do Citoesqueleto/genética , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica , RNA Longo não Codificante/genética , Transdução de Sinais/fisiologia , Proteínas Correpressoras/metabolismo , Proteínas do Citoesqueleto/metabolismo , RNA Helicases DEAD-box/metabolismo , Células HCT116 , Células HeLa , Humanos , RNA Longo não Codificante/metabolismo , Fase S , Regulação para CimaRESUMO
Differentiation status of tumors is correlated with metastatic potential and malignancy. FOXA1 (forkhead box A1) is a transcription factor known to regulate differentiation in certain tissues. Here, we investigate FOXA1 function in human colorectal cancer (CRC). We found that FOXA1 is robustly expressed in the normal human colon but significantly downregulated in colon adenocarcinoma. Applying FOXA1 chromatin immunoprecipitation coupled with deep sequencing and transcriptome analysis upon FOXA1 knockdown in well-differentiated CRC cells and FOXA1 overexpression in poorly differentiated CRC cells, we identified novel protein-coding and lncRNA genes regulated by FOXA1. Among the numerous novel FOXA1 targets we identified, we focused on CEACAM5, a tumor marker and facilitator of cell adhesion. We show that FOXA1 binds to a distal enhancer downstream of CEACAM5 and strongly activates its expression. Consistent with these data, we show that FOXA1 inhibits anoikis in CRC cells. Collectively, our results uncover novel protein-coding and noncoding targets of FOXA1 and suggest a vital role of FOXA1 in enhancing CEACAM5 expression and anoikis resistance in CRC cells.
Assuntos
Neoplasias Colorretais/genética , Redes Reguladoras de Genes , Fator 3-alfa Nuclear de Hepatócito/genética , RNA Longo não Codificante/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Anoikis/genética , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Neoplasias Colorretais/patologia , Elementos Facilitadores Genéticos , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação Neoplásica da Expressão Gênica , Estudo de Associação Genômica Ampla , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas/genética , PseudogenesRESUMO
When breast cancer progresses to a metastatic stage, survival rates decline rapidly and it is considered incurable. Thus, deciphering the critical mechanisms of metastasis is of vital importance to develop new treatment options. We hypothesize that studying the proteins that are newly synthesized during the metastatic processes of migration and invasion will greatly enhance our understanding of breast cancer progression. We conducted a mass spectrometry screen following bioorthogonal noncanonical amino acid tagging to elucidate changes in the nascent proteome that occur during epidermal growth factor stimulation in migrating and invading cells. Annexin A2 was identified in this screen and subsequent examination of breast cancer cell lines revealed that Annexin A2 is specifically upregulated in estrogen receptor negative (ER-) cell lines. Furthermore, siRNA knockdown showed that Annexin A2 expression promotes the proliferation, wound healing and directional migration of breast cancer cells. In patients, Annexin A2 expression is increased in ER- breast cancer subtypes. Additionally, high Annexin A2 expression confers a higher probability of distant metastasis specifically for ER- patients. This work establishes a pivotal role of Annexin A2 in breast cancer progression and identifies Annexin A2 as a potential therapeutic target for the more aggressive and harder to treat ER- subtype.
Assuntos
Anexina A2/metabolismo , Neoplasias da Mama/metabolismo , Anexina A2/genética , Western Blotting , Neoplasias da Mama/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Imunoprecipitação , Células MCF-7 , Espectrometria de Massas , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismoRESUMO
Cellular quiescence and cell cycle reentry regulate vital biological processes such as cellular development and tissue homeostasis and are controlled by precise regulation of gene expression. The roles of long noncoding RNAs (lncRNAs) during these processes remain to be elucidated. By performing genome-wide transcriptome analyses, we identify differential expression of several hundreds of lncRNAs, including a significant number of the less-characterized class of microRNA-host-gene (MIRHG) lncRNAs or lnc-MIRHGs, during cellular quiescence and cell cycle reentry in human diploid fibroblasts. We observe that MIR222HG lncRNA displays serum-stimulated RNA processing due to enhanced splicing of the host nascent pri-MIR222HG transcript. The pre-mRNA splicing factor SRSF1 negatively regulates the microprocessor-catalyzed cleavage of pri-miR-222, thereby increasing the cellular pool of the mature MIR222HG Association of SRSF1 to pri-MIR222HG, including to a mini-exon, which partially overlaps with the primary miR-222 precursor, promotes serum-stimulated splicing over microRNA processing of MIR222HG Further, we observe that the increased levels of spliced MIR222HG in serum-stimulated cells promote the cell cycle reentry post quiescence in a microRNA-independent manner. MIR222HG interacts with DNM3OS, another lncRNA whose expression is elevated upon serum-stimulation, and promotes cell cycle reentry. The double-stranded RNA binding protein ILF3/2 complex facilitates MIR222HG:DNM3OS RNP complex assembly, thereby promoting DNM3OS RNA stability. Our study identifies a novel mechanism whereby competition between the splicing and microprocessor machinery modulates the serum-induced RNA processing of MIR222HG, which dictates cell cycle reentry.
Assuntos
Perfilação da Expressão Gênica/métodos , Pulmão/citologia , RNA Longo não Codificante/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Soro/química , Ciclo Celular , Linhagem Celular , Fibroblastos/química , Fibroblastos/citologia , Células HEK293 , Humanos , Pulmão/química , Proteína do Fator Nuclear 45/metabolismo , Proteínas do Fator Nuclear 90/metabolismo , Processamento Pós-Transcricional do RNA , Splicing de RNA , Análise de Sequência de RNA , Imagem Individual de Molécula , Regulação para Cima , Sequenciamento do ExomaRESUMO
Circular RNAs (circRNAs) are a class of noncoding RNAs produced by a noncanonical form of alternative splicing called back-splicing. To investigate a potential role of circRNAs in the p53 pathway, we analyzed RNA sequencing (RNA-seq) data from colorectal cancer cell lines (HCT116, RKO, and SW48) that were untreated or treated with a DNA-damaging agent. Surprisingly, unlike the strong p53-dependent induction of hundreds of p53-induced mRNAs upon DNA damage, only a few circRNAs were upregulated from p53-induced genes. circ-MDM2, an annotated circRNA from the MDM2 locus, was one of the handful of circRNAs that originated from a p53-induced gene. Given the central role of MDM2 in suppressing p53 protein levels and p53 activity, we investigated the function of circ-MDM2 Knocking down circ-MDM2 with small interfering RNAs (siRNAs) that targeted circ-MDM2 did not alter MDM2 mRNA or MDM2 protein levels but resulted in increased basal p53 levels and growth defects in vitro and in vivo Consistent with these results, transcriptome profiling showed increased expression of several direct p53 targets, reduced retinoblastoma protein (Rb) phosphorylation, and defects in G1-S progression upon silencing circ-MDM2 Our results on the initial characterization of circ-MDM2 identify a new player from the MDM2 locus that suppresses p53 levels and cell cycle progression.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2/genética , RNA Circular/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ciclo Celular/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Dano ao DNA , Perfilação da Expressão Gênica/métodos , Células HCT116 , Humanos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA/metabolismo , RNA Circular/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Análise de Sequência de RNA/métodos , Proteína Supressora de Tumor p53/genéticaRESUMO
Recent advancements in genetic and proteomic technologies have revealed that more of the genome encodes proteins than originally thought possible. Specifically, some putative long noncoding RNAs (lncRNAs) have been misannotated as noncoding. Numerous lncRNAs have been found to contain short open reading frames (sORFs) which have been overlooked because of their small size. Many of these sORFs encode small proteins or micropeptides with fundamental biological importance. These micropeptides can aid in diverse processes, including cell division, transcription regulation, and cell signaling. Here we discuss strategies for establishing the coding potential of putative lncRNAs and describe various functions of known micropeptides.