RESUMO
Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.
Assuntos
Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , MicroRNAs/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Receptores de Estrogênio/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Sequências Repetidas Invertidas/genética , Sequências Repetidas Invertidas/fisiologia , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , MicroRNAs/genética , Proteínas Associadas à Matriz Nuclear/genética , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores de Estrogênio/genéticaRESUMO
LIN28 is a bipartite RNA-binding protein that post-transcriptionally inhibits the biogenesis of let-7 microRNAs to regulate development and influence disease states. However, the mechanisms of let-7 suppression remain poorly understood because LIN28 recognition depends on coordinated targeting by both the zinc knuckle domain (ZKD), which binds a GGAG-like element in the precursor, and the cold shock domain (CSD), whose binding sites have not been systematically characterized. By leveraging single-nucleotide-resolution mapping of LIN28 binding sites in vivo, we determined that the CSD recognizes a (U)GAU motif. This motif partitions the let-7 microRNAs into two subclasses, precursors with both CSD and ZKD binding sites (CSD+) and precursors with ZKD but no CSD binding sites (CSD-). LIN28 in vivo recognition-and subsequent 3' uridylation and degradation-of CSD+ precursors is more efficient, leading to their stronger suppression in LIN28-activated cells and cancers. Thus, CSD binding sites amplify the regulatory effects of LIN28.
Assuntos
MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Células-Tronco Embrionárias , Células Hep G2 , Humanos , Células K562 , Camundongos , MicroRNAs/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Domínios Proteicos , Estrutura Terciária de Proteína , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Environmental cues provoke rapid transitions in gene expression to support growth and cellular plasticity through incompletely understood mechanisms. Lin28 RNA-binding proteins have evolutionarily conserved roles in post-transcriptional coordination of pro-growth gene expression, but signaling pathways allowing trophic stimuli to induce Lin28 have remained uncharacterized. We find that Lin28a protein exhibits rapid basal turnover in neurons and that mitogen-activated protein kinase (MAPK)-dependent phosphorylation of the RNA-silencing factor HIV TAR-RNA-binding protein (TRBP) promotes binding and stabilization of Lin28a, but not Lin28b, with an accompanying reduction in Lin28-regulated miRNAs, downstream of brain-derived neurotrophic factor (BDNF). Binding of Lin28a to TRBP in vitro is also enhanced by phospho-mimic TRBP. Further, phospho-TRBP recapitulates BDNF-induced neuronal dendritic spine growth in a Lin28a-dependent manner. Finally, we demonstrate MAPK-dependent TRBP and Lin28a induction, with physiological function in growth and survival, downstream of diverse growth factors in multiple primary cell types, supporting a broad role for this pathway in trophic responses.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Espinhas Dendríticas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proliferação de Células , Sobrevivência Celular , Células HEK293 , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Humanos , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/metabolismo , Camundongos , Neurônios/metabolismo , FosforilaçãoRESUMO
During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify â¼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis.
Assuntos
MicroRNAs/biossíntese , Precursores de RNA/biossíntese , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Células A549 , Sítios de Ligação , Sistemas CRISPR-Cas , RNA Helicases DEAD-box/metabolismo , Bases de Dados Genéticas , Regulação da Expressão Gênica , Genômica/métodos , Células HEK293 , Células HeLa , Células Hep G2 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células Jurkat , Células MCF-7 , MicroRNAs/química , MicroRNAs/genética , Conformação de Ácido Nucleico , Ligação Proteica , Proteômica/métodos , Interferência de RNA , Precursores de RNA/química , Precursores de RNA/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/metabolismo , Análise de Sequência de RNA , Relação Estrutura-Atividade , Transfecção , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: The maturation of microRNAs (miRNAs) successively undergoes Drosha, Dicer, and Argonaute -mediated processing, however, the intricate regulations of the individual miRNA maturation are largely unknown. Retinoid x receptor alpha (RXRα) belongs to nuclear receptors that regulate gene transcription by binding to DNA elements, however, whether RXRα binds to miRNAs to exert physiological functions is not known. RESULTS: In this work, we found that RXRα directly binds to the precursor of miR-103 (pre-miR-103a-2) via its DNA-binding domain with a preferred binding sequence of AGGUCA. The binding of RXRα inhibits the processing of miR-103 maturation from pre-miR-103a-2. Mechanistically, RXRα prevents the nuclear export of pre-miR-103a-2 for further processing by inhibiting the association of exportin-5 with pre-miR-103a-2. Pathophysiologically, the negative effect of RXRα on miR-103 maturation correlates to the positive effects of RXRα on the expression of Dicer, a target of miR-103, and on the inhibition of breast cancer. CONCLUSIONS: Our findings unravel an unexpected role of transcription factor RXRα in specific miRNA maturation at post-transcriptional level through pre-miRNA binding, and present a mechanistic insight regarding RXRα role in breast cancer progression.
Assuntos
MicroRNAs , Receptores Citoplasmáticos e Nucleares , Fatores de Transcrição , Proteínas Argonautas , MicroRNAs/genéticaRESUMO
Dicer-like 1 (DCL1) is a core component of the plant microRNA (miRNA) biogenesis machinery. MiRNA is transcribed as a precursor RNA, termed primary miRNA (pri-miRNA), which is cleaved by DCL1 in two steps to generate miRNA/miRNA* duplex. Pri-miRNA is a single-stranded RNA that forms a hairpin structure with a number of unpaired bases, hereafter called mismatches, on its stem. In the present study, by using purified recombinant Arabidopsis DCL1, we presented evidence that mismatches on the stem of pri-miRNA are important for precise DCL1 cleavage. We showed that a mismatch at the loop-distal side of the end of miRNA/miRNA* duplex is important for efficient cleavage of pri-miRNA in vitro, as previously suggested in planta. On the contrary, mismatches distant from the miRNA/miRNA* duplex region are important for determining the cleavage position by DCL1. The purified DCL1 proteins cleaved mutant pri-miRNA variants without such mismatches at a position at which wild-type pri-miRNA variants are not usually cleaved, resulting in an increased accumulation of small RNA different from miRNA. Therefore, our results suggest that, in addition to the distance from the ssRNA-dsRNA junction, mismatches on the stem of pri-miRNA function as a determinant for precise processing of pri-miRNA by DCL1 in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Ciclo Celular , MicroRNAs , Ribonuclease III , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Nucleotídeos/metabolismo , Processamento Pós-Transcricional do RNA , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
MicroRNAs (miRNAs) are small noncoding RNAs that function as critical posttranscriptional regulators in various biological processes. While most miRNAs are generated from processing of long primary transcripts via sequential Drosha and Dicer cleavage, some miRNAs that bypass Drosha cleavage can be transcribed as part of another small noncoding RNA. Here, we develop the target-oriented miRNA discovery (TOMiD) bioinformatic analysis method to identify Drosha-independent miRNAs from Argonaute crosslinking and sequencing of hybrids (Ago-CLASH) data sets. Using this technique, we discovered a novel miRNA derived from a primate specific noncoding RNA, the small NF90 associated RNA A (snaR-A). The miRNA derived from snaR-A (miR-snaR) arises independently of Drosha processing but requires Exportin-5 and Dicer for biogenesis. We identify that miR-snaR is concurrently up-regulated with the full snaR-A transcript in cancer cells. Functionally, miR-snaR associates with Ago proteins and targets NME1, a key metastasis inhibitor, contributing to snaR-A's role in promoting cancer cell migration. Our findings suggest a functional link between a novel miRNA and its precursor noncoding RNA.
Assuntos
Biologia Computacional/métodos , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , Regiões 3' não Traduzidas , Movimento Celular , RNA Helicases DEAD-box/metabolismo , Células HCT116 , Células HEK293 , Humanos , Carioferinas/metabolismo , Células MCF-7 , Nucleosídeo NM23 Difosfato Quinases/antagonistas & inibidores , Nucleosídeo NM23 Difosfato Quinases/genética , Neoplasias/patologia , RNA Longo não Codificante/metabolismo , Ribonuclease III/metabolismoRESUMO
Herpesvirus saimiri (HVS) is an oncogenic γ-herpesvirus that produces microRNAs (miRNAs) by cotranscription of precursor miRNA (pre-miRNA) hairpins immediately downstream from viral small nuclear RNAs (snRNA). The host cell Integrator complex, which recognizes the snRNA 3' end processing signal (3' box), generates the 5' ends of HVS pre-miRNA hairpins. Here, we identify a novel 3' box-like sequence (miRNA 3' box) downstream from HVS pre-miRNAs that is essential for miRNA biogenesis. In vivo knockdown and rescue experiments confirmed that the 3' end processing of HVS pre-miRNAs also depends on Integrator activity. Interaction between Integrator and HVS primary miRNA (pri-miRNA) substrates that contain only the miRNA 3' box was confirmed by coimmunoprecipitation and an in situ proximity ligation assay (PLA) that we developed to localize specific transient RNA-protein interactions inside cells. Surprisingly, in contrast to snRNA 3' end processing, HVS pre-miRNA 3' end processing by Integrator can be uncoupled from transcription, enabling new approaches to study Integrator enzymology.
Assuntos
Herpesvirus Saimiriíneo 2/genética , MicroRNAs/metabolismo , Processamento de Terminações 3' de RNA/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Herpesvirus Saimiriíneo 2/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Imunoprecipitação , MicroRNAs/química , MicroRNAs/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Processamento de Terminações 3' de RNA/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Nuclear Pequeno/metabolismo , Transcrição GênicaRESUMO
Polo-Like Kinase 1 (PLK1), a key mediator of cell-cycle progression, is associated with poor prognosis and is a therapeutic target in a number of malignancies. Putative phosphorylation sites for PLK1 have been identified on Drosha, the main catalytic component of the microprocessor responsible for miR biogenesis. Several kinases, including GSK3ß, p70 S6 kinase, ABL, PAK5, p38 MAPK, CSNK1A1 and ANKRD52-PPP6C, have been shown to phosphorylate components of the miR biogenesis machinery, altering their activity and/or localisation, and therefore the biogenesis of distinct miR subsets. We hypothesised that PLK1 regulates miR biogenesis through Drosha phosphorylation. In vitro kinase assays confirmed PLK1 phosphorylation of Drosha at S300 and/or S302. PLK1 inhibition reduced serine-phosphorylated levels of Drosha and its RNA-dependent association with DGCR8. In contrast, a "phospho-mimic" Drosha mutant showed increased association with DGCR8. PLK1 phosphorylation of Drosha alters Drosha Microprocessor complex subcellular localisation, since PLK1 inhibition increased cytosolic protein levels of both DGCR8 and Drosha, whilst nuclear levels were decreased. Importantly, the above effects are independent of PLK1's cell cycle-regulatory role, since altered Drosha:DGCR8 localisation upon PLK1 inhibition occurred prior to significant accumulation of cells in M-phase, and PLK1-regulated miRs were not increased in M-phase-arrested cells. Small RNA sequencing and qPCR validation were used to assess downstream consequences of PLK1 activity on miR biogenesis, identifying a set of ten miRs (miR-1248, miR-1306-5p, miR-2277-5p, miR-29c-5p, miR-93-3p, miR-152-3p, miR-509-3-5p, miR-511-5p, miR-891a-5p and miR-892a) whose expression levels were statistically significantly downregulated by two pharmacological PLK1 kinase domain inhibitors, RO-5203280 and GSK461364. Opposingly, increased levels of these miRs were observed upon transfection of wild-type or constitutively active PLK1. Importantly, pre-miR levels were reduced upon PLK1 inhibition, and pri-miR levels decreased upon PLK1 activation, and hence, PLK1 Drosha phosphorylation regulates MiR biogenesis at the level of pri-miR-to-pre-miR processing. In combination with prior studies, this work identifies Drosha S300 and S302 as major integration points for signalling by several kinases, whose relative activities will determine the relative biogenesis efficiency of different miR subsets. Identified kinase-regulated miRs have potential for use as kinase inhibitor response-predictive biomarkers, in cancer and other diseases.
Assuntos
MicroRNAs , MicroRNAs/genética , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas de Ligação a RNA , Quinase 1 Polo-LikeRESUMO
MicroRNAs are small noncoding RNAs that regulate gene expression; stabilize the cell phenotype; and play an important role in cell differentiation, development, and apoptosis. A canonical microRNA biogenesis pathway includes several posttranscriptional steps of processing and transport and ends with cytoplasmic cleavage of pre-miRNA by type III ribonuclease DICER to form a mature duplex, which is included in RISC. MicroRNA biogenesis and role in cell stress are still poorly understood. Using flow cytometry and high-throughput analysis of gene expression, we have shown that chronic endoplasmic reticulum (ER) stress, which is associated with improper protein folding in the ER, induce a cellular senescence phenotype in fibroblast-like FRSN cells. While acute ER stress can reduce miRNA biogenesis, chronic stress does not cause a significant drop in global microRNA expression and is accompanied by only a slight decrease in DICER1 mRNA expression. Heterogeneity with respect to lysosomal ß-galactosidase activity was found to increase in the cell population exposed to ER stress. We do not exclude induced cell heterogeneity regarding expression of components of the microRNA biogenesis pathway.
Assuntos
MicroRNAs , MicroRNAs/metabolismo , Estresse do Retículo Endoplasmático/genética , Senescência Celular/genética , ApoptoseRESUMO
Convincing evidence has shown that microRNAs (miRNAs) are involved in the pathogenesis of stroke. This study aimed to examine whether miRNA biogenesis genes polymorphisms are associated with risk of large artery atherosclerosis (LAA) stroke. Three polymorphisms (DROSHA rs10719 T>C, RAN rs3803012 A>G, and PIWIL1 rs10773771 C>T) were screened by certain criteria. A total of 1,785 (710 cases and 1,075 controls) study subjects were included in this study. We found that rs10773771 CC genotype was associated with a decreased risk of LAA stroke (CC vs. TT/CT: OR 0.63, 95% CI 0.46-0.86, P = 3 × 10-3). In silico analysis suggested that rs10773771 can change the mRNA secondary structure of PIWIL1 and affect the binding of the miRNAs and regulatory motifs to the 3'-UTR of PIWIL1. Expression quantitative trait loci analysis showed that rs10773771 could change the expression of PIWIL1 in human skin (P = 1.534 × 10-10) and thyroid tissues (P = 4.869 × 10-6). These findings suggested that PIWIL1 rs10773771 may be associated with a decreased risk of LAA stroke.
Assuntos
Proteínas Argonautas , Aterosclerose , MicroRNAs , Acidente Vascular Cerebral , Regiões 3' não Traduzidas , Proteínas Argonautas/genética , Artérias/metabolismo , Artérias/patologia , Aterosclerose/complicações , Aterosclerose/genética , Aterosclerose/patologia , Estudos de Casos e Controles , Predisposição Genética para Doença , Genótipo , Humanos , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Acidente Vascular Cerebral/complicaçõesRESUMO
The contributions of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) to breast cancer are critical areas of investigation. In this study, we identified a novel lncRNA RP11-283G6.5 which was lowly expressed in breast cancer and whose low expression was correlated with poor overall survival and disease-free survival of breast cancer patients. Functional experiments revealed that ectopic expression of RP11-283G6.5 confined breast cancer cellular growth, migration, and invasion, and promoted cellular apoptosis. Conversely, RP11-283G6.5 silencing facilitated breast cancer cellular growth, migration, and invasion, and repressed cellular apoptosis. Moreover, RP11-283G6.5 was found to confine breast cancer tumour growth and metastasis in vivo. Mechanistically, RP11-283G6.5 competitively bound to ILF3, reduced the binding of ILF3to primary miR-188 (pri-miR-188), abolished the suppressive effect of ILF3 on pri-miR-188 processing, and therefore promoted pri-miR-188 processing, leading to the reduction of pri-miR-188 and the upregulation of mature miR-188-3p. The expression of RP11-283G6.5 was significantly positively correlated with that of miR-188-3p in breast cancer tissues. Through increasing miR-188-3p, RP11-283G6.5 decreased TMED3, a target of miR-188-3p. RP11-283G6.5 further suppressed Wnt/ß-catenin signalling via decreasing TMED3. Rescue assays revealed that inhibition of miR-188-3p, overexpression of TMED3 or blocking Wnt/ß-catenin signalling all attenuated the roles of RP11-283G6.5 in breast cancer. Collectively, these findings demonstrated that RP11-283G6.5 is a tumour suppressive lncRNA in breast cancer via modulating miR-188-3p/TMED3/Wnt/ß-catenin signalling. This study indicated that RP11-283G6.5 might be a promising prognostic biomarker and therapeutic target for breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , RNA Longo não Codificante/genética , Proteínas de Transporte Vesicular/metabolismo , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas , Proteínas de Transporte Vesicular/genética , Proteína Wnt1/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genéticaRESUMO
MicroRNAs are posttranscriptional regulators of gene expression. As microRNAs can target many genes simultaneously, microRNAs can regulate complex multifactorial processes, including post-ischemic neovascularization, a major recovery pathway in cardiovascular disease. MicroRNAs select their target mRNAs via full complementary binding with their seed sequence, i.e., nucleotides 2-8 from the 5' end of a microRNA. The exact sequence of a mature microRNA, and thus of its 5' and 3' ends, is determined by two sequential cleavage steps of microRNA precursors, Drosha/DGCR8 and Dicer. When these cleavage steps result in nucleotide switches at the 5' end, forming a so-called 5'-isomiR, this results in a shift in the mature microRNA's seed sequence. The role of 5'-isomiRs in cardiovascular diseases is still unknown. Here, we characterize the expression and function of the 5'-isomiR of miR-411 (ISO-miR-411). ISO-miR-411 is abundantly expressed in human primary vascular cells. ISO-miR-411 has a different "targetome" from WT-miR-411, with only minor overlap. The ISO-miR-411/WT-miR-411 ratio is downregulated under acute ischemia, both in cells and a murine ischemia model, but is upregulated instead in chronically ischemic human blood vessels. ISO-miR-411 negatively influences vascular cell migration, whereas WT-miR-411 does not. Our data demonstrate that isomiR formation is a functional pathway that is actively regulated during ischemia.
Assuntos
Endotélio Vascular/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Isquemia/genética , MicroRNAs/genética , Neovascularização Fisiológica/genética , Animais , Sequência de Bases , Movimento Celular/genética , Células Cultivadas , RNA Helicases DEAD-box/genética , Bases de Dados Genéticas , Modelos Animais de Doenças , Membro Posterior/irrigação sanguínea , Membro Posterior/patologia , Humanos , Extremidade Inferior/irrigação sanguínea , Extremidade Inferior/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença Arterial Periférica/patologia , Ribonuclease III/genéticaRESUMO
By 2003, the Human Genome project had been completed; however, it turned out that 97% of genome sequences did not encode proteins. The explanation came later when it was found the untranslated DNA contain sequences for short microRNAs (miRNAs) and long noncoding RNAs that did not produce any mRNAs or tRNAs, but instead were involved in the regulation of gene expression. Initially identified in the cytoplasm, miRNAs have been found in all cell compartments, where their functions are not limited to the degradation of target mRNAs. miRNAs that are secreted into the extracellular space as components of exosomes or as complexes with proteins, participate in morphogenesis, regeneration, oncogenesis, metastasis, and chemoresistance of tumor cells. miRNAs play a dual role in oncogenesis: on one hand, they act as oncogene suppressors; on the other hand, they function as oncogenes themselves and inactivate oncosuppressors, stimulate tumor neoangiogenesis, and mediate immunosuppressive processes in the tumors, The review presents current concepts of the miRNA biogenesis and their functions in the cytoplasm and nucleus with special focus on the noncanonical mechanisms of gene regulation by miRNAs and involvement of miRNAs in oncogenesis, as well as the authors' opinion on the role of miRNAs in metastasis and formation of the premetastatic niche.
Assuntos
MicroRNAs/metabolismo , Neoplasias/metabolismo , Exossomos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genéticaRESUMO
Vitrification of embryos has been known as the most efficient cryopreservation method in assisted reproductive technology clinics. Vitrification of preimplantation embryo might be associated with altered gene expression profile and biochemical changes of vitrified embryos. Stringent regulation of gene expression in early embryonic stages is very critical for normal development. In the present study, we investigated the effect of vitrification on the canonical miRNA biogenesis pathway, and also the expression of developmental related miRNAs, in 8-cell and blastocyst mouse embryos. Although the expression pattern of the miRNA biogenesis pathway genes differed between 8-cell and blastocyst mouse embryos, vitrification did not affect the expression level of these genes in preimplantation embryos. The expression levels of miR-21 and let-7a were significantly decreased in vitrified 8-cell embryos and fresh blastocysts when compared with fresh 8-cell embryos. The expression of Stat3 was significantly reduced in blastocysts after vitrification. The alteration in the expression pattern of miRNAs, due to their mode of action, can affect broad downstream key developmental signaling pathways. Therefore, the blastocyst stage is the preferred point for embryo vitrification as they are less susceptible to cryo-damages regarding the stability of miRNAs related to the developmental and implantation competence of embryo.
Assuntos
Vitrificação , Animais , Blastocisto , Criopreservação , Desenvolvimento Embrionário/genética , Camundongos , MicroRNAs/genéticaRESUMO
MicroRNAs (miRNAs) are small, non-coding RNAs that function as endogenous gene silencers. Soon after the discovery of miRNAs, a subset of brain-enriched and brain-specific miRNAs were identified and significant advancements were made in delineating miRNA function in brain development. However, understanding the molecular mechanisms that regulate miRNA biogenesis in normal and diseased brains has become a prevailing challenge. Besides transcriptional regulation of miRNA host genes, miRNA processing intermediates are subjected to multifaceted regulation by canonical miRNA processing enzymes, RNA binding proteins (RBPs) and epitranscriptomic modifications. Further still, miRNA activity can be regulated by the sponging activity of other non-coding RNA classes, namely circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs). Differential abundance of these factors in neuronal and glial lineages partly underlies the spatiotemporal expression and function of lineage-specific miRNAs. Here, we review the continuously evolving understanding of the regulation of neuronal and glial miRNA biogenesis at the transcriptional and posttranscriptional levels and the cooperativity of miRNA species in targeting key mRNAs to drive lineage-specific development. In addition, we review dysregulation of neuronal and glial miRNAs and the detrimental impacts which contribute to developmental brain disorders.
Assuntos
Encefalopatias/patologia , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Neuroglia/patologia , Neurônios/patologia , RNA Mensageiro/metabolismo , Animais , Encefalopatias/genética , Encefalopatias/metabolismo , Humanos , Neuroglia/metabolismo , Neurônios/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genéticaRESUMO
RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.
Assuntos
RNA Helicases DEAD-box/fisiologia , Células Endoteliais/fisiologia , Neovascularização Fisiológica , Ribonuclease III/fisiologia , Animais , HumanosRESUMO
MicroRNAs (miRNAs or miRs) are small, noncoding RNAs that are implicated in the regulation of most biological processes. Global miRNA biogenesis is altered in many cancers, and RNA-binding proteins play a role in miRNA biogenesis, presenting a promising avenue for targeting miRNA dysregulation in diseases. miR-34a exhibits tumor-suppressive activities by targeting cell cycle regulators CDK4/6 and anti-apoptotic factor BCL-2, among other regulatory pathways such as Wnt, TGF-ß, and Notch signaling. Many cancers exhibit down-regulation or loss of miR-34a, and synthetic miR-34a supplementation has been shown to inhibit tumor growth in vivo However, the post-transcriptional mechanisms that cause miR-34a loss in cancer are not entirely understood. Here, using a proteomics-mediated approach in non-small-cell lung cancer (NSCLC) cells, we identified squamous cell carcinoma antigen recognized by T-cells 3 (SART3) as a putative pre-miR-34a-binding protein. SART3 is a spliceosome recycling factor and nuclear RNA-binding protein with no previously reported role in miRNA regulation. We found that SART3 binds pre-miR-34a with higher specificity than pre-let-7d (used as a negative control) and elucidated a new functional role for SART3 in NSCLC cells. SART3 overexpression increased miR-34a levels, down-regulated the miR-34a target genes CDK4/6, and caused a cell cycle arrest in the G1 phase. In vitro binding experiments revealed that the RNA-recognition motifs within the SART3 sequence are responsible for selective pre-miR-34a binding. Our results provide evidence for a significant role of SART3 in miR-34a biogenesis and cell cycle progression in NSCLC cells.
Assuntos
Antígenos de Neoplasias/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Proteínas de Neoplasias/genética , Proteínas de Ligação a RNA/genética , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Ligação Proteica/genética , Proteômica/métodos , Spliceossomos/genéticaRESUMO
Altered gene expression is the primary molecular mechanism responsible for the pathological processes of human diseases, including cancer. MicroRNAs (miRNAs) are virtually involved at the post-transcriptional level and bind to 3' UTR of their target messenger RNA (mRNA) to suppress expression. Dysfunction of miRNAs disturbs expression of oncogenic or tumor-suppressive target genes, which is implicated in cancer pathogenesis. As such, a large number of miRNAs have been found to be downregulated or upregulated in human cancers and to function as oncomiRs or oncosuppressor miRs. Notably, the molecular mechanism underlying the dysregulation of miRNA expression in cancer has been recently uncovered. The genetic deletion or amplification and epigenetic methylation of miRNA genomic loci and the transcription factor-mediated regulation of primary miRNA often alter the landscape of miRNA expression in cancer. Dysregulation of the multiple processing steps in mature miRNA biogenesis can also cause alterations in miRNA expression in cancer. Detailed knowledge of the regulatory mechanism of miRNAs in cancer is essential for understanding its physiological role and the implications of cancer-associated dysfunction and dysregulation. In this review, we elucidate how miRNA expression is deregulated in cancer, paying particular attention to the cancer-associated transcriptional and post-transcriptional factors that execute miRNA programs.
Assuntos
MicroRNAs/metabolismo , Neoplasias/genética , Animais , Epigênese Genética/genética , Epigênese Genética/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , MicroRNAs/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
MicroRNAs (miRNAs) play a key role in fine-tuning host immune homeostasis and responses through the negative regulation of mRNA stability and translation. The pathways regulated by miRNAs are well characterized, but the precise mechanisms that control the miRNA-mediated regulation of gene expression during immune cell-development and immune responses to invading pathogens are incompletely understood. Context-specific interactions of miRNAs with other RNA species or proteins may modulate the function of a given miRNA. Dysregulation of miRNA function is associated with various human diseases, such as cardiovascular diseases and cancers. Here, we review the potential modulators of miRNA function in the immune system, including the transcription regulators of miRNA genes, miRNA-processing enzymes, factors affecting miRNA targeting, and intercellular communication.