RESUMO
Super-enhancers are an emerging subclass of regulatory regions controlling cell identity and disease genes. However, their biological function and impact on miRNA networks are unclear. Here, we report that super-enhancers drive the biogenesis of master miRNAs crucial for cell identity by enhancing both transcription and Drosha/DGCR8-mediated primary miRNA (pri-miRNA) processing. Super-enhancers, together with broad H3K4me3 domains, shape a tissue-specific and evolutionarily conserved atlas of miRNA expression and function. CRISPR/Cas9 genomics revealed that super-enhancer constituents act cooperatively and facilitate Drosha/DGCR8 recruitment and pri-miRNA processing to boost cell-specific miRNA production. The BET-bromodomain inhibitor JQ1 preferentially inhibits super-enhancer-directed cotranscriptional pri-miRNA processing. Furthermore, super-enhancers are characterized by pervasive interaction with DGCR8/Drosha and DGCR8/Drosha-regulated mRNA stability control, suggesting unique RNA regulation at super-enhancers. Finally, super-enhancers mark multiple miRNAs associated with cancer hallmarks. This study presents principles underlying miRNA biology in health and disease and an unrecognized higher-order property of super-enhancers in RNA processing beyond transcription.
Assuntos
Elementos Facilitadores Genéticos , MicroRNAs/metabolismo , Animais , Azepinas/farmacologia , Regulação da Expressão Gênica , Código das Histonas , Humanos , Camundongos , Neoplasias/genética , Especificidade de Órgãos , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Transcrição Gênica , Triazóis/farmacologiaRESUMO
Microprocessor (MP), DROSHA-DGCR8, processes primary miRNA transcripts (pri-miRNAs) to initiate miRNA biogenesis. The canonical cleavage mechanism of MP has been extensively investigated and comprehensively validated for two decades. However, this canonical mechanism cannot account for the processing of certain pri-miRNAs in animals. In this study, by conducting high-throughput pri-miRNA cleavage assays for approximately 260,000 pri-miRNA sequences, we discovered and comprehensively characterized a noncanonical cleavage mechanism of MP. This noncanonical mechanism does not need several RNA and protein elements essential for the canonical mechanism; instead, it utilizes previously unrecognized DROSHA dsRNA recognition sites (DRESs). Interestingly, the noncanonical mechanism is conserved across animals and plays a particularly significant role in C. elegans. Our established noncanonical mechanism elucidates MP cleavage in numerous RNA substrates unaccounted for by the canonical mechanism in animals. This study suggests a broader substrate repertoire of animal MPs and an expanded regulatory landscape for miRNA biogenesis.
Assuntos
MicroRNAs , Animais , MicroRNAs/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismo , RNA de Cadeia Dupla , Processamento Pós-Transcricional do RNARESUMO
DROSHA serves as a gatekeeper of the microRNA (miRNA) pathway by processing primary transcripts (pri-miRNAs). While the functions of structured domains of DROSHA have been well documented, the contribution of N-terminal proline-rich disordered domain (PRD) remains elusive. Here we show that the PRD promotes the processing of miRNA hairpins located within introns. We identified a DROSHA isoform (p140) lacking the PRD, which is produced by proteolytic cleavage. Small RNA sequencing revealed that p140 is significantly impaired in the maturation of intronic miRNAs. Consistently, our minigene constructs demonstrated that PRD enhances the processing of intronic hairpins, but not those in exons. Splice site mutations did not affect the PRD's enhancing effect on intronic constructs, suggesting that the PRD acts independently of splicing reaction by interacting with sequences residing within introns. The N-terminal regions from zebrafish and Xenopus DROSHA can replace the human counterpart, indicating functional conservation despite poor sequence alignment. Moreover, we found that rapidly evolving intronic miRNAs are generally more dependent on PRD than conserved ones, suggesting a role of PRD in miRNA evolution. Our study reveals a new layer of miRNA regulation mediated by a low-complexity disordered domain that senses the genomic contexts of miRNA loci.
Assuntos
MicroRNAs , Ribonuclease III , Animais , Humanos , Íntrons/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Prolina/genética , Prolina/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Processamento Pós-Transcricional do RNA , Peixe-ZebraRESUMO
Maturation of canonical microRNA (miRNA) is initiated by DROSHA that cleaves the primary transcript (pri-miRNA). More than 1,800 miRNA loci are annotated in humans, but it remains largely unknown whether and at which sites pri-miRNAs are cleaved by DROSHA. Here, we performed in vitro processing on a full set of human pri-miRNAs (miRBase version 21) followed by sequencing. This comprehensive profiling enabled us to classify miRNAs on the basis of DROSHA dependence and map their cleavage sites with respective processing efficiency measures. Only 758 pri-miRNAs are confidently processed by DROSHA, while the majority may be non-canonical or false entries. Analyses of the DROSHA-dependent pri-miRNAs show key cis-elements for processing. We observe widespread alternative processing and unproductive cleavage events such as "nick" or "inverse" processing. SRSF3 is a broad-acting auxiliary factor modulating alternative processing and suppressing unproductive processing. The profiling data and methods developed in this study will allow systematic analyses of miRNA regulation.
Assuntos
MicroRNAs/genética , Processamento Pós-Transcricional do RNA/genética , Ribonuclease III/genética , Fatores de Processamento de Serina-Arginina/genética , Sítios de Ligação/genética , Genoma Humano/genética , Células HEK293 , Humanos , Interferência de RNARESUMO
Nuclear processing of most miRNAs is mediated by Microprocessor, comprised of RNase III enzyme Drosha and its cofactor DGCR8. Here, we uncover a hidden layer of Microprocessor regulation via studies of Dicer-independent mir-451, which is clustered with canonical mir-144. Although mir-451 is fully dependent on Drosha/DGCR8, its short stem and small terminal loop render it an intrinsically weak Microprocessor substrate. Thus, it must reside within a cluster for normal biogenesis, although the identity and orientation of its neighbor are flexible. We use DGCR8 tethering assays and operon structure-function assays to demonstrate that local recruitment and transfer of Microprocessor enhances suboptimal substrate processing. This principle applies more broadly since genomic analysis indicates suboptimal canonical miRNAs are enriched in operons, and we validate several of these experimentally. Proximity-based enhancement of suboptimal hairpin processing provides a rationale for genomic retention of certain miRNA operons and may explain preferential evolutionary emergence of miRNA operons.
Assuntos
Genômica , MicroRNAs/genética , Proteínas de Ligação a RNA/genética , Ribonuclease III/genética , Núcleo Celular/genética , Humanos , Processamento Pós-Transcricional do RNA/genéticaRESUMO
A commencing and critical step in miRNA biogenesis involves processing of pri-miRNAs in the nucleus by Microprocessor. An important, but not completely understood, question is how Drosha, the catalytic subunit of Microprocessor, binds pri-miRNAs and correctly specifies cleavage sites. Here we report the cryoelectron microscopy structures of the Drosha-DGCR8 complex with and without a pri-miRNA. The RNA-bound structure provides direct visualization of the tertiary structure of pri-miRNA and shows that a helix hairpin in the extended PAZ domain and the mobile basic (MB) helix in the RNase IIIa domain of Drosha coordinate to recognize the single-stranded to double-stranded junction of RNA, whereas the dsRNA binding domain makes extensive contacts with the RNA stem. Furthermore, the RNA-free structure reveals an autoinhibitory conformation of the PAZ helix hairpin. These findings provide mechanistic insights into pri-miRNA cleavage site selection and conformational dynamics governing pri-miRNA recognition by the catalytic component of Microprocessor.
Assuntos
MicroRNAs/química , MicroRNAs/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Animais , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Spodoptera/citologiaRESUMO
Primary microRNAs (miRNAs) are the precursors of miRNAs that modulate the expression of most mRNAs in humans. They fold up into a hairpin structure that is cleaved at its base by an enzyme complex known as the Microprocessor (Drosha/DGCR8). While many of the molecular details are known, a complete understanding of what features distinguish primary miRNA from hairpin structures in other transcripts is still lacking. We develop a massively parallel functional assay termed Dro-seq (Drosha sequencing) that enables testing of hundreds of known primary miRNA substrates and thousands of single-nucleotide variants. We find an additional feature of primary miRNAs, called Shannon entropy, describing the structural ensemble important for processing. In a deep mutagenesis experiment, we observe particular apical loop U bases, likely recognized by DGCR8, are important for efficient processing. These findings build on existing knowledge about primary miRNA maturation by the Microprocessor and further explore the substrate RNA sequence-structure relationship.
Assuntos
MicroRNAs , Complexos Multiproteicos , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA , Ribonuclease III , Animais , Humanos , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/química , Ribonuclease III/metabolismo , Células Sf9 , SpodopteraRESUMO
Microprocessor initiates the processing of microRNAs (miRNAs) from the hairpin regions of primary transcripts (pri-miRNAs). Pri-miRNAs often contain multiple miRNA hairpins, and this clustered arrangement can assist in the processing of otherwise defective hairpins. We find that miR-451, which derives from a hairpin with a suboptimal terminal loop and a suboptimal stem length, accumulates to 40-fold higher levels when clustered with a helper hairpin. This phenomenon tolerates changes in hairpin order, linker lengths, and the identities of the helper hairpin, the recipient hairpin, the linker-sequence, and the RNA polymerase that transcribes the hairpins. It can act reciprocally and need not occur co-transcriptionally. It requires Microprocessor recognition of the helper hairpin and linkage of the two hairpins, yet predominantly manifests after helper-hairpin processing. It also requires enhancer of rudimentary homolog (ERH), which copurifies with Microprocessor and can dimerize and interact with other proteins that can dimerize, suggesting a model in which one Microprocessor recruits another Microprocessor.
Assuntos
Proteínas de Ciclo Celular/genética , MicroRNAs/genética , RNA Polimerase III/genética , Fatores de Transcrição/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/genética , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição GênicaRESUMO
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
Metazoan microRNAs require specific maturation steps initiated by Microprocessor, comprising Drosha and DGCR8. Lack of structural information for the assembled complex has hindered an understanding of how Microprocessor recognizes primary microRNA transcripts (pri-miRNAs). Here we present a cryoelectron microscopy structure of human Microprocessor with a pri-miRNA docked in the active site, poised for cleavage. The basal junction is recognized by a four-way intramolecular junction in Drosha, triggered by the Belt and Wedge regions that clamp over the ssRNA. The belt is important for efficiency and accuracy of pri-miRNA processing. Two dsRBDs form a molecular ruler to measure the stem length between the two dsRNA-ssRNA junctions. The specific organization of the dsRBDs near the apical junction is independent of Drosha core domains, as observed in a second structure in the partially docked state. Collectively, we derive a molecular model to explain how Microprocessor recognizes a pri-miRNA and accurately identifies the cleavage site.
Assuntos
MicroRNAs/química , Proteínas de Ligação a RNA/química , Ribonuclease III/química , Microscopia Crioeletrônica , Humanos , MicroRNAs/metabolismo , Modelos Moleculares , Conformação Proteica , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/metabolismoRESUMO
Microprocessor, composed of DROSHA and its cofactor DGCR8, initiates microRNA (miRNA) biogenesis by processing the primary transcripts of miRNA (pri-miRNAs). Here we investigate the mechanism by which Microprocessor selects the cleavage site with single-nucleotide precision, which is crucial for the specificity and functionality of miRNAs. By testing â¼40,000 pri-miRNA variants, we find that for some pri-miRNAs the cleavage site is dictated mainly by the mGHG motif embedded in the lower stem region of pri-miRNA. Structural modeling and deep-sequencing-based complementation experiments show that the double-stranded RNA-binding domain (dsRBD) of DROSHA recognizes mGHG to place the catalytic center in the appropriate position. The mGHG motif as well as the mGHG-recognizing residues in DROSHA dsRBD are conserved across eumetazoans, suggesting that this mechanism emerged in an early ancestor of the animal lineage. Our findings provide a basis for the understanding of miRNA biogenesis and rational design of accurate small-RNA-based gene silencing.
Assuntos
MicroRNAs/metabolismo , Motivos de Nucleotídeos , Processamento Pós-Transcricional do RNA , Ribonuclease III/metabolismo , Células HCT116 , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MicroRNAs/química , MicroRNAs/genética , Modelos Moleculares , Conformação de Ácido Nucleico , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The microRNAs encoded by the miR-17â¼92 polycistron are commonly overexpressed in cancer and orchestrate a wide range of oncogenic functions. Here, we identify a mechanism for miR-17â¼92 oncogenic function through the disruption of endogenous microRNA (miRNA) processing. We show that, upon oncogenic overexpression of the miR-17â¼92 primary transcript (pri-miR-17â¼92), the microprocessor complex remains associated with partially processed intermediates that aberrantly accumulate. These intermediates reflect a series of hierarchical and conserved steps in the early processing of the pri-miR-17â¼92 transcript. Encumbrance of the microprocessor by miR-17â¼92 intermediates leads to the broad but selective downregulation of co-expressed polycistronic miRNAs, including miRNAs derived from tumor-suppressive miR-34b/c and from the Dlk1-Dio3 polycistrons. We propose that the identified steps of polycistronic miR-17â¼92 biogenesis contribute to the oncogenic re-wiring of gene regulation networks. Our results reveal previously unappreciated functional paradigms for polycistronic miRNAs in cancer.
Assuntos
Carcinogênese/genética , MicroRNAs/genética , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação ao Cálcio/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Iodeto Peroxidase/genética , Proteínas de Membrana/genética , MicroRNAs/biossíntese , Conformação de Ácido NucleicoRESUMO
The nuclear cleavage of a suboptimal primary miRNA hairpin by the Drosha/DGCR8 complex ("Microprocessor") can be enhanced by an optimal miRNA neighbor, a phenomenon termed cluster assistance. Several features and biological impacts of this new layer of miRNA regulation are not fully known. Here, we elucidate the parameters of cluster assistance of a suboptimal miRNA and also reveal competitive interactions amongst optimal miRNAs within a cluster. We exploit cluster assistance as a functional assay for suboptimal processing and use this to invalidate putative suboptimal substrates, as well as identify a "solo" suboptimal miRNA. Finally, we report complexity in how specific mutations might affect the biogenesis of clustered miRNAs in disease contexts. This includes how an operon context can buffer the effect of a deleterious processing variant, but reciprocally how a point mutation can have a nonautonomous effect to impair the biogenesis of a clustered, suboptimal, neighbor. These data expand our knowledge regarding regulated miRNA biogenesis in humans and represent a functional assay for empirical definition of suboptimal Microprocessor substrates.
Assuntos
MicroRNAs , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
The potential for microRNAs (miRNAs) to regulate gene expression remains incompletely understood. DROSHA initiates the biogenesis of miRNAs while variants of Argonaute (AGO) and trinucleotide repeat containing six (TNRC6) family proteins form complexes with miRNAs to facilitate RNA recognition and gene regulation. Here we investigate the fate of miRNAs in the absence of these critical RNAi protein factors. Knockout of DROSHA expression reduces levels of some miRNAs annotated in miRBase but not others. The identity of miRNAs with reduced expression matches the identity of miRNAs previously identified by experimental approaches. The MirGeneDB resource offers the closest alignment with experimental results. In contrast, the loss of TNRC6 proteins had much smaller effects on miRNA levels. Knocking out AGO proteins, which directly contact the mature miRNA, decreased expression of the miRNAs most strongly associated with AGO2 as determined from enhanced crosslinking immunoprecipitation (AGO2-eCLIP). Evaluation of miRNA binding to endogenously expressed AGO proteins revealed that miRNA:AGO association was similar for AGO1, AGO2, AGO3, and AGO4. Our data emphasize the need to evaluate annotated miRNAs based on approximate cellular abundance, DROSHA-dependence, and physical association with AGO when forming hypotheses related to their function.
Assuntos
MicroRNAs , MicroRNAs/metabolismo , Interferência de RNA , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Regulação da Expressão Gênica , Repetições de TrinucleotídeosRESUMO
MicroRNA (miRNA) maturation is initiated by DROSHA, a double-stranded RNA (dsRNA)-specific RNase III enzyme. By cleaving primary miRNAs (pri-miRNAs) at specific positions, DROSHA serves as a main determinant of miRNA sequences and a highly selective gatekeeper for the canonical miRNA pathway. However, the sites of DROSHA-mediated processing have not been annotated, and it remains unclear to what extent DROSHA functions outside the miRNA pathway. Here, we establish a protocol termed "formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq)," which allows identification of DROSHA cleavage sites at single-nucleotide resolution. fCLIP identifies numerous processing sites, suggesting widespread end modifications during miRNA maturation. fCLIP also finds many pri-miRNAs that undergo alternative processing, yielding multiple miRNA isoforms. Moreover, we discovered dozens of DROSHA substrates on non-miRNA loci, which may serve as cis-elements for DROSHA-mediated gene regulation. We anticipate that fCLIP-seq could be a general tool for investigating interactions between dsRNA-binding proteins and structured RNAs.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Ribonuclease III/metabolismo , Análise de Sequência de RNA/métodos , Sequência de Bases , Sítios de Ligação , Reagentes de Ligações Cruzadas/química , Formaldeído/química , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , MicroRNAs/química , MicroRNAs/genética , Conformação de Ácido Nucleico , Ligação Proteica , Interferência de RNA , Ribonuclease III/química , Ribonuclease III/genética , Relação Estrutura-Atividade , Especificidade por Substrato , TransfecçãoRESUMO
MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression and play critical roles in tumorigenesis. Genetic variants in miRNA processing genes, DROSHA and DICER, have been implicated in cancer susceptibility and progression in various populations. However, their role in Egyptian patients with breast cancer (BC) remains unexplored. This study aims to investigate the association of DROSHA rs10719 and DICER rs3742330 polymorphisms with BC risk and clinical outcomes. This case-control study included 209 BC patients and 106 healthy controls. Genotyping was performed using TaqMan assays in blood, tumor tissue, and adjacent non-cancerous tissue samples. Associations were analyzed using logistic regression and Fisher's exact test. The DROSHA rs10719 AA genotype was associated with a 3.2-fold increased risk (95%CI = 1.23-9.36, p < 0.001), and the DICER rs3742330 GG genotype was associated with a 3.51-fold increased risk (95%CI = 1.5-8.25, p = 0.001) of BC. Minor allele frequencies were 0.42 for rs10719 A and 0.37 for rs3742330 G alleles. The risk alleles were significantly more prevalent in tumor tissue than adjacent normal tissue (rs10719 A: 40.8% vs. 0%; rs3742330 G: 42.7% vs. 0%; p < 0.001). However, no significant associations were observed with clinicopathological features or survival outcomes over a median follow-up of 17 months. In conclusion, DROSHA rs10719 and DICER rs3742330 polymorphisms are associated with increased BC risk and more prevalent in tumor tissue among our cohort, suggesting a potential role in miRNA dysregulation during breast tumorigenesis. These findings highlight the importance of miRNA processing gene variants in BC susceptibility and warrant further validation in larger cohorts and different ethnic populations.
RESUMO
BACKGROUND: Excessive fatty acids in the liver lead to the accumulation of lipotoxic lipids and then cellular stress to further evoke the related disease, like non-alcoholic fatty liver disease (NAFLD). As reported, fatty acid stimulation can cause some specific miRNA dysregulation, which caused us to investigate the relationship between miRNA biogenesis and fatty acid overload. METHODS: Gene expression omnibus (GEO) dataset analysis, miRNA-seq, miRNA cleavage assay, RT-qPCR, western blotting, immunofluorescence and co-immunoprecipitation (co-IP) were used to reveal the change of miRNAs under pathological status and explore the relevant mechanism. High fat, high fructose, high cholesterol (HFHFrHC) diet-fed mice transfected with AAV2/8-shDrosha or AAV2/8-shPRMT5 were established to investigate the in vivo effects of Drosha or PRMT5 on NAFLD phenotype. RESULTS: We discovered that the cleavage of miRNAs was inhibited by analysing miRNA contents and detecting some representative pri-miRNAs in multiple mouse and cell models, which was further verified by the reduction of the Microprocessor activity in the presence of palmitic acid (PA). In vitro, PA could induce Drosha, the core RNase III in the Microprocessor complex, degrading through the proteasome-mediated pathway, while in vivo, knockdown of Drosha significantly promoted NAFLD to develop to a more serious stage. Mechanistically, our results demonstrated that PA can increase the methyltransferase activity of PRMT5 to degrade Drosha through MDM2, a ubiquitin E3 ligase for Drosha. The above results indicated that PRMT5 may be a critical regulator in lipid metabolism during NAFLD, which was confirmed by the knocking down of PRMT5 improved aberrant lipid metabolism in vitro and in vivo. CONCLUSIONS: We first demonstrated the relationship between miRNA dosage and NAFLD and proved that PA can activate the PRMT5-MDM2-Drosha signalling pathway to regulate miRNA biogenesis.
Assuntos
Metabolismo dos Lipídeos , MicroRNAs , Hepatopatia Gordurosa não Alcoólica , Proteína-Arginina N-Metiltransferases , Proteínas Proto-Oncogênicas c-mdm2 , Animais , Humanos , Masculino , Camundongos , Dieta Hiperlipídica , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Fígado/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , MicroRNAs/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Ribonuclease III/metabolismo , Ribonuclease III/genética , Transdução de SinaisRESUMO
Recent studies suggest that the microprocessor (Drosha-DGCR8) complex can be recruited to chromatin to catalyze co-transcriptional processing of primary microRNAs (pri-miRNAs) in mammalian cells. However, the molecular mechanism of co-transcriptional miRNA processing is poorly understood. Here we find that HP1BP3, a histone H1-like chromatin protein, specifically associates with the microprocessor and promotes global miRNA biogenesis in human cells. Chromatin immunoprecipitation (ChIP) studies reveal genome-wide co-localization of HP1BP3 and Drosha and HP1BP3-dependent Drosha binding to actively transcribed miRNA loci. Moreover, HP1BP3 specifically binds endogenous pri-miRNAs and facilitates the Drosha/pri-miRNA association in vivo. Knockdown of HP1BP3 compromises pri-miRNA processing by causing premature release of pri-miRNAs from the chromatin. Taken together, these studies suggest that HP1BP3 promotes co-transcriptional miRNA processing via chromatin retention of nascent pri-miRNA transcripts. This work significantly expands the functional repertoire of the H1 family of proteins and suggests the existence of chromatin retention factors for widespread co-transcriptional miRNA processing.
Assuntos
Cromatina/metabolismo , MicroRNAs/biossíntese , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , Transcrição Gênica , Animais , Sítios de Ligação , Cromatina/genética , Imunoprecipitação da Cromatina , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Proteínas de Ligação a DNA , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genoma Humano , Células HeLa , Humanos , MicroRNAs/genética , Proteínas Nucleares/genética , Ligação Proteica , Interferência de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , TransfecçãoRESUMO
Mirtrons represent a subclass of microRNAs (miRNAs) that rely on the splicing machinery for their maturation. However, the molecular details of this Drosha-independent processing are still not fully understood; as an example, the Microprocessor complex cannot process the mirtronic pre-miRNA from the transcript even if splice site mutations are present. To investigate the influence of alternative splicing sites on mirtron formation, we generated Enhanced Green Fluorescent Protein (EGFP) reporters containing artificial introns to compare the processing of canonical miRNAs and mirtrons. Although mutations of both splice sites generated a complex pattern of alternative transcripts, mirtron formation was always severely affected as opposed to the normal processing of the canonical hsa-mir-33b miRNA. However, we also detected that while its formation was also hindered, the mirtron-derived hsa-mir-877-3p miRNA was less affected by certain mutations than the hsa-mir-877-5p species. By knocking down Drosha, we showed that this phenomenon is not dependent on Microprocessor activity but rather points toward the potential stability difference between the miRNAs from the different arms. Our results indicate that when the major splice sites are mutated, mirtron formation cannot be rescued by nearby alternative splice sites, and stability differences between 5p and 3p species should also be considered for functional studies of mirtrons.
Assuntos
Processamento Alternativo , MicroRNAs , Ribonuclease III , MicroRNAs/genética , Humanos , Ribonuclease III/genética , Ribonuclease III/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , Mutação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Íntrons/genéticaRESUMO
The DNA damage response (DDR) is the signaling cascade that recognizes DNA double-strand breaks (DSBs) and promotes their resolution via the DNA repair pathways of non-homologous end joining (NHEJ) or homologous recombination (HR). We and others have shown that DDR activation requires DROSHA; however, whether DROSHA exerts its functions by associating with damage sites, what controls its recruitment, and how DROSHA influences DNA repair remains poorly understood. Here, we show that DROSHA associates with DSBs independently of transcription. Neither H2AX, nor ATM or DNA-PK kinase activities are required for recruitment of DROSHA to break sites. Rather, DROSHA interacts with RAD50, and inhibition of the MRN complex by mirin treatment abolishes this interaction. MRN complex inactivation by RAD50 knockdown or mirin treatment prevents DROSHA recruitment to DSBs and, as a consequence, also prevents 53BP1 (also known as TP53BP1) recruitment. During DNA repair, DROSHA inactivation reduces NHEJ and boosts HR frequency. Indeed, DROSHA knockdown also increases the association of downstream HR factors such as RAD51 to DNA ends. Overall, our results demonstrate that DROSHA is recruited at DSBs by the MRN complex and directs DNA repair towards NHEJ.