RESUMEN
Cellular senescence is an irreversible state of cell-cycle arrest induced by various stresses, including aberrant oncogene activation, telomere shortening, and DNA damage. Through a genome-wide screen, we discovered a conserved small nucleolar RNA (snoRNA), SNORA13, that is required for multiple forms of senescence in human cells and mice. Although SNORA13 guides the pseudouridylation of a conserved nucleotide in the ribosomal decoding center, loss of this snoRNA minimally impacts translation. Instead, we found that SNORA13 negatively regulates ribosome biogenesis. Senescence-inducing stress perturbs ribosome biogenesis, resulting in the accumulation of free ribosomal proteins (RPs) that trigger p53 activation. SNORA13 interacts directly with RPL23, decreasing its incorporation into maturing 60S subunits and, consequently, increasing the pool of free RPs, thereby promoting p53-mediated senescence. Thus, SNORA13 regulates ribosome biogenesis and the p53 pathway through a non-canonical mechanism distinct from its role in guiding RNA modification. These findings expand our understanding of snoRNA functions and their roles in cellular signaling.
Asunto(s)
Senescencia Celular , ARN Nucleolar Pequeño , Proteínas Ribosómicas , Ribosomas , Proteína p53 Supresora de Tumor , Humanos , ARN Nucleolar Pequeño/metabolismo , ARN Nucleolar Pequeño/genética , Senescencia Celular/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Ribosomas/metabolismo , Animales , Ratones , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genéticaRESUMEN
Long noncoding RNAs (lncRNAs) have emerged as regulators of diverse biological processes. Here, we describe the initial functional analysis of a poorly characterized human lncRNA (LINC00657) that is induced after DNA damage, which we termed "noncoding RNA activated by DNA damage", or NORAD. NORAD is highly conserved and abundant, with expression levels of approximately 500-1,000 copies per cell. Remarkably, inactivation of NORAD triggers dramatic aneuploidy in previously karyotypically stable cell lines. NORAD maintains genomic stability by sequestering PUMILIO proteins, which repress the stability and translation of mRNAs to which they bind. In the absence of NORAD, PUMILIO proteins drive chromosomal instability by hyperactively repressing mitotic, DNA repair, and DNA replication factors. These findings introduce a mechanism that regulates the activity of a deeply conserved and highly dosage-sensitive family of RNA binding proteins and reveal unanticipated roles for a lncRNA and PUMILIO proteins in the maintenance of genomic stability.
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Inestabilidad Genómica , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Secuencia de Bases , Inestabilidad Cromosómica , Células HCT116 , Humanos , Ratones , Ploidias , ARN Largo no Codificante/química , ARN Largo no Codificante/genéticaRESUMEN
Although splicing is a major driver of RNA nuclear export, many intronless RNAs are efficiently exported to the cytoplasm through poorly characterized mechanisms. For example, GC-rich sequences promote nuclear export in a splicing-independent manner, but how GC content is recognized and coupled to nuclear export is unknown. Here, we developed a genome-wide screening strategy to investigate the mechanism of export of NORAD, an intronless cytoplasmic long noncoding RNA (lncRNA). This screen revealed an RNA binding protein, RBM33, that directs the nuclear export of NORAD and numerous other transcripts. RBM33 directly binds substrate transcripts and recruits components of the TREX-NXF1/NXT1 RNA export pathway. Interestingly, high GC content emerged as the feature that specifies RBM33-dependent nuclear export. Accordingly, RBM33 directly binds GC-rich elements in target transcripts. These results provide a broadly applicable strategy for the genetic dissection of nuclear export mechanisms and reveal a long-sought nuclear export pathway for transcripts with GC-rich sequences.
Asunto(s)
Proteínas de Transporte Nucleocitoplasmático , ARN Viral , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte de ARN , ARN Viral/metabolismoRESUMEN
A Retraction to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Change history: In this Letter, the citation to 'Fig. 4e, f' in the main text should be 'Fig. 3e, f'. This has not been corrected online.
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Therapeutic strategies based on modulation of microRNA (miRNA) activity hold great promise due to the ability of these small RNAs to potently influence cellular behavior. In this study, we investigated the efficacy of a miRNA replacement therapy for liver cancer. We demonstrate that hepatocellular carcinoma (HCC) cells exhibit reduced expression of miR-26a, a miRNA that is normally expressed at high levels in diverse tissues. Expression of this miRNA in liver cancer cells in vitro induces cell-cycle arrest associated with direct targeting of cyclins D2 and E2. Systemic administration of this miRNA in a mouse model of HCC using adeno-associated virus (AAV) results in inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and dramatic protection from disease progression without toxicity. These findings suggest that delivery of miRNAs that are highly expressed and therefore tolerated in normal tissues but lost in disease cells may provide a general strategy for miRNA replacement therapies.
Asunto(s)
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/terapia , MicroARNs/uso terapéutico , Animales , Ciclina D2 , Ciclinas/metabolismo , Dependovirus/genética , Modelos Animales de Enfermedad , Vectores Genéticos , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.
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Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Silenciador del Gen , MicroARNs/genética , Secuencia de Aminoácidos , Proteínas Argonautas/química , Sistemas CRISPR-Cas/genética , Quinasa de la Caseína II/metabolismo , Células HCT116 , Humanos , MicroARNs/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por SustratoRESUMEN
Bone-resorbing osteoclasts significantly contribute to osteoporosis and bone metastases of cancer. MicroRNAs play important roles in physiology and disease, and present tremendous therapeutic potential. Nonetheless, how microRNAs regulate skeletal biology is underexplored. Here we identify miR-34a as a novel and critical suppressor of osteoclastogenesis, bone resorption and the bone metastatic niche. miR-34a is downregulated during osteoclast differentiation. Osteoclastic miR-34a-overexpressing transgenic mice exhibit lower bone resorption and higher bone mass. Conversely, miR-34a knockout and heterozygous mice exhibit elevated bone resorption and reduced bone mass. Consequently, ovariectomy-induced osteoporosis, as well as bone metastasis of breast and skin cancers, are diminished in osteoclastic miR-34a transgenic mice, and can be effectively attenuated by miR-34a nanoparticle treatment. Mechanistically, we identify transforming growth factor-ß-induced factor 2 (Tgif2) as an essential direct miR-34a target that is pro-osteoclastogenic. Tgif2 deletion reduces bone resorption and abolishes miR-34a regulation. Together, using mouse genetic, pharmacological and disease models, we reveal miR-34a as a key osteoclast suppressor and a potential therapeutic strategy to confer skeletal protection and ameliorate bone metastasis of cancers.
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Neoplasias Óseas/prevención & control , Neoplasias Óseas/secundario , Diferenciación Celular/genética , MicroARNs/genética , Osteoclastos/patología , Osteoporosis/prevención & control , Proteínas Represoras/deficiencia , Animales , Secuencia de Bases , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , Neoplasias Mamarias Animales/patología , Ratones , Ratones Transgénicos , MicroARNs/farmacología , MicroARNs/uso terapéutico , Trasplante de Neoplasias , Tamaño de los Órganos/efectos de los fármacos , Osteoclastos/efectos de los fármacos , Osteoporosis/genética , Osteoporosis/patología , Ovariectomía , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Neoplasias Cutáneas/patología , Transgenes , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Precise regulation of microRNA (miRNA) expression is critical for diverse physiologic and pathophysiologic processes. Nevertheless, elucidation of the mechanisms through which miRNA expression is regulated has been greatly hindered by the incomplete annotation of primary miRNA (pri-miRNA) transcripts. While a subset of miRNAs are hosted in protein-coding genes, the majority of pri-miRNAs are transcribed as poorly characterized noncoding RNAs that are 10's to 100's of kilobases in length and low in abundance due to efficient processing by the endoribonuclease DROSHA, which initiates miRNA biogenesis. Accordingly, these transcripts are poorly represented in existing RNA-seq data sets and exhibit limited and inaccurate annotation in current transcriptome assemblies. To overcome these challenges, we developed an experimental and computational approach that allows genome-wide detection and mapping of pri-miRNA structures. Deep RNA-seq in cells expressing dominant-negative DROSHA resulted in much greater coverage of pri-miRNA transcripts compared with standard RNA-seq. A computational pipeline was developed that produces highly accurate pri-miRNA assemblies, as confirmed by extensive validation. This approach was applied to a panel of human and mouse cell lines, providing pri-miRNA transcript structures for 1291/1871 human and 888/1181 mouse miRNAs, including 594 human and 425 mouse miRNAs that fall outside protein-coding genes. These new assemblies uncovered unanticipated features and new potential regulatory mechanisms, including links between pri-miRNAs and distant protein-coding genes, alternative pri-miRNA splicing, and transcripts carrying subsets of miRNAs encoded by polycistronic clusters. These results dramatically expand our understanding of the organization of miRNA-encoding genes and provide a valuable resource for the study of mammalian miRNA regulation.
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Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , MicroARNs/química , MicroARNs/genética , Anotación de Secuencia Molecular , Conformación de Ácido Nucleico , Empalme Alternativo , Evolución Molecular , Estudio de Asociación del Genoma Completo/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , MicroARNs/metabolismo , Familia de Multigenes , Sistemas de Lectura Abierta , Regiones Promotoras Genéticas , ARN no Traducido , Ribonucleasa III/metabolismo , Análisis de Secuencia de ARN , Transcripción GenéticaRESUMEN
Altered glucose metabolism in cancer cells is termed the Warburg effect, which describes the propensity of most cancer cells to take up glucose avidly and convert it primarily to lactate, despite available oxygen. Notwithstanding the renewed interest in the Warburg effect, cancer cells also depend on continued mitochondrial function for metabolism, specifically glutaminolysis that catabolizes glutamine to generate ATP and lactate. Glutamine, which is highly transported into proliferating cells, is a major source of energy and nitrogen for biosynthesis, and a carbon substrate for anabolic processes in cancer cells, but the regulation of glutamine metabolism is not well understood. Here we report that the c-Myc (hereafter referred to as Myc) oncogenic transcription factor, which is known to regulate microRNAs and stimulate cell proliferation, transcriptionally represses miR-23a and miR-23b, resulting in greater expression of their target protein, mitochondrial glutaminase, in human P-493 B lymphoma cells and PC3 prostate cancer cells. This leads to upregulation of glutamine catabolism. Glutaminase converts glutamine to glutamate, which is further catabolized through the tricarboxylic acid cycle for the production of ATP or serves as substrate for glutathione synthesis. The unique means by which Myc regulates glutaminase uncovers a previously unsuspected link between Myc regulation of miRNAs, glutamine metabolism, and energy and reactive oxygen species homeostasis.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Glutaminasa/metabolismo , Glutamina/metabolismo , MicroARNs/metabolismo , Mitocondrias/enzimología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Regiones no Traducidas 3'/metabolismo , Línea Celular Tumoral , HumanosRESUMEN
CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Through a genome-wide CRISPR screen, we identify protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout breast cancer cells. Inhibition of PRMT5 blocks the G1-to-S transition in the cell cycle independent of RB, leading to growth arrest in RB1-knockout cells. Proteomics analysis uncovers fused in sarcoma (FUS) as a downstream effector of PRMT5. Inhibition of PRMT5 results in dissociation of FUS from RNA polymerase II, leading to hyperphosphorylation of serine 2 in RNA polymerase II, intron retention, and subsequent downregulation of proteins involved in DNA synthesis. Furthermore, treatment with the PRMT5 inhibitor pemrametostat and a selective ER degrader fulvestrant synergistically inhibits growth of ER+/RB-deficient cell-derived and patient-derived xenografts. These findings highlight dual ER and PRMT5 blockade as a potential therapeutic strategy to overcome resistance to CDK4/6i in ER+/RB-deficient breast cancer.
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Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , ARN Polimerasa II , Quinasa 4 Dependiente de la Ciclina/metabolismo , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Resistencia a Antineoplásicos/genética , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismoRESUMEN
During translation of the genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus in the COVID-19 pandemic, host ribosomes undergo programmed ribosomal frameshifting (PRF) at a conserved structural element. Although PRF is essential for coronavirus replication, host factors that regulate this process have not yet been identified. Here we perform genome-wide CRISPR-Cas9 knockout screens to identify regulators of SARS-CoV-2 PRF. These screens reveal that loss of ribosome recycling factors markedly decreases frameshifting efficiency and impairs SARS-CoV-2 viral replication. Mutational studies support a model wherein efficient removal of ribosomal subunits at the ORF1a stop codon is required for frameshifting of trailing ribosomes. This dependency upon ribosome recycling is not observed with other non-pathogenic human betacoronaviruses and is likely due to the unique position of the ORF1a stop codon in the SARS clade of coronaviruses. These findings therefore uncover host factors that support efficient SARS-CoV-2 translation and replication.
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COVID-19 , Sistema de Lectura Ribosómico , Humanos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , COVID-19/metabolismo , Codón de Terminación/genética , Codón de Terminación/metabolismo , Pandemias , Replicación Viral/genética , Ribosomas/metabolismo , ARN Viral/metabolismoRESUMEN
Increased nucleolar size and activity correlate with aberrant ribosome biogenesis and enhanced translation in cancer cells. One of the first and rate-limiting steps in translation is the interaction of the 40S small ribosome subunit with mRNAs. Here, we report the identification of the zinc finger protein 692 (ZNF692), a MYC-induced nucleolar scaffold that coordinates the final steps in the biogenesis of the small ribosome subunit. ZNF692 forms a hub containing the exosome complex and ribosome biogenesis factors specialized in the final steps of 18S rRNA processing and 40S ribosome maturation in the granular component of the nucleolus. Highly proliferative cells are more reliant on ZNF692 than normal cells; thus, we conclude that effective production of small ribosome subunits is critical for translation efficiency in cancer cells.
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Proteínas de Unión al ADN , Biosíntesis de Proteínas , Proteínas Ribosómicas , Subunidades Ribosómicas Pequeñas de Eucariotas , Factores de Transcripción , Nucléolo Celular/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , Humanos , Animales , Ratas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with estrogen receptor-positive (ER+) breast cancer. However, patients treated with CDK4/6i eventually develop drug resistance and progress. RB1 loss-of-function alterations confer acquired resistance to CDK4/6i, but the optimal therapy for these patients is unclear. Using a genome-wide CRISPR screen, we identified protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in ER+/RB1-knockout (RBKO) breast cancer cells. PRMT5 inhibition blocked cell cycle G1-to-S transition independent of RB, thus arresting growth of RBKO cells. Proteomics analysis uncovered fused in sarcoma (FUS) as a downstream effector of PRMT5. Pharmacological inhibition of PRMT5 resulted in dissociation of FUS from RNA polymerase II (Pol II), Ser2 Pol II hyperphosphorylation, and intron retention in genes that promote DNA synthesis. Treatment with the PRMT5i inhibitor pemrametostat and fulvestrant synergistically inhibited growth of ER+/RB-deficient patient-derived xenografts, suggesting dual ER and PRMT5 blockade as a novel therapeutic strategy to treat ER+/RB-deficient breast cancer.
RESUMEN
Direct control of microRNA (miRNA) expression by oncogenic and tumor suppressor networks results in frequent dysregulation of miRNAs in cancer cells and contributes to tumorigenesis. We previously demonstrated that activation of the c-Myc oncogenic transcription factor (Myc) broadly influences miRNA expression and in particular leads to widespread miRNA down-regulation. miRNA transcripts repressed by Myc include several with potent tumor suppressor activity such as miR-15a/16-1, miR-34a, and let-7 family members. In this study, we have investigated mechanisms downstream of Myc that contribute to miRNA repression. Consistent with transcriptional down-regulation, Myc activity results in the decreased abundance of multiple miRNA primary transcripts. Surprisingly, however, primary transcripts encoding several let-7 miRNAs are not reduced in the high Myc state, suggesting a posttranscriptional mechanism of repression. The Lin-28 and Lin-28B RNA binding proteins were recently demonstrated to negatively regulate let-7 biogenesis. We now show that Myc induces Lin-28B expression in multiple human and mouse tumor models. Chromatin immunoprecipitation and reporter assays reveal direct association of Myc with the Lin-28B promoter resulting in transcriptional transactivation. Moreover, we document that activation of Lin-28B is necessary and sufficient for Myc-mediated let-7 repression. Accordingly, Lin-28B loss-of-function significantly impairs Myc-dependent cellular proliferation. These findings highlight an important role for Lin-28B in Myc-driven cellular phenotypes and uncover an orchestration of transcriptional and posttranscriptional mechanisms in Myc-mediated reprogramming of miRNA expression.
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Regulación hacia Abajo/genética , MicroARNs/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Activación Transcripcional/genética , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genéticaRESUMEN
In mammalian cells, the enzymatic pathways involved in cytoplasmic mRNA decay are incompletely defined. In this study, we have used two approaches to disrupt activities of deadenylating and/or decapping enzymes to monitor effects on mRNA decay kinetics and trap decay intermediates. Our results show that deadenylation is the key first step that triggers decay of both wild-type stable and nonsense codon-containing unstable beta-globin mRNAs in mouse NIH3T3 fibroblasts. PAN2 and CCR4 are the major poly(A) nucleases active in cytoplasmic deadenylation that have biphasic kinetics, with PAN2 initiating deadenylation followed by CCR4-mediated poly(A) shortening. DCP2-mediated decapping takes place after deadenylation and may serve as a backup mechanism for triggering mRNA decay when initial deadenylation by PAN2 is compromised. Our findings reveal a functional link between deadenylation and decapping and help to define in vivo pathways for mammalian cytoplasmic mRNA decay.
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Endorribonucleasas/metabolismo , Exorribonucleasas/metabolismo , Estabilidad del ARN/genética , ARN Mensajero/metabolismo , Animales , Núcleo Celular/enzimología , Citoplasma/metabolismo , Endorribonucleasas/análisis , Endorribonucleasas/genética , Exorribonucleasas/análisis , Exorribonucleasas/genética , Fibroblastos/enzimología , Globinas/genética , Humanos , Ratones , Células 3T3 NIH , Interferencia de ARNRESUMEN
Sequences within 5' UTRs dictate the site and efficiency of translation initiation. In this study, an unbiased screen designed to interrogate the 5' UTR-mediated regulation of the growth-promoting gene MYC unexpectedly revealed the ribosomal pause relief factor eIF5A as a regulator of translation initiation codon selection. Depletion of eIF5A enhances upstream translation within 5' UTRs across yeast and human transcriptomes, including on the MYC transcript, where this results in increased production of an N-terminally extended protein. Furthermore, ribosome profiling experiments established that the function of eIF5A as a suppressor of ribosomal pausing at sites of suboptimal peptide bond formation is conserved in human cells. We present evidence that proximal ribosomal pausing on a transcript triggers enhanced use of upstream suboptimal or non-canonical initiation codons. Thus, we propose that eIF5A functions not only to maintain efficient translation elongation in eukaryotic cells but also to maintain the fidelity of translation initiation.
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Codón Iniciador/genética , Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN/genética , Ribosomas/metabolismo , Regiones no Traducidas 5'/genética , Línea Celular , Línea Celular Tumoral , Células HCT116 , Células HEK293 , Humanos , Extensión de la Cadena Peptídica de Translación/genética , Biosíntesis de Proteínas/genética , Proteínas Proto-Oncogénicas c-myc/genética , Factor 5A Eucariótico de Iniciación de TraducciónRESUMEN
NORAD is a conserved long noncoding RNA (lncRNA) that is required for genome stability in mammals. NORAD acts as a negative regulator of PUMILIO (PUM) proteins in the cytoplasm, and we previously showed that loss of NORAD or PUM hyperactivity results in genome instability and premature aging in mice (Kopp et al., 2019). Recently, however, it was reported that NORAD regulates genome stability through an interaction with the RNA binding protein RBMX in the nucleus. Here, we addressed the contributions of NORAD:PUM and NORAD:RBMX interactions to genome maintenance by this lncRNA in human cells. Extensive RNA FISH and fractionation experiments established that NORAD localizes predominantly to the cytoplasm with or without DNA damage. Moreover, genetic rescue experiments demonstrated that PUM binding is required for maintenance of genomic stability by NORAD whereas binding of RBMX is dispensable for this function. These data provide an important foundation for further mechanistic dissection of the NORAD-PUMILIO axis in genome maintenance.
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Inestabilidad Genómica , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Línea Celular , Humanos , Unión Proteica , Mapas de Interacción de ProteínasRESUMEN
MicroRNAs (miRNAs) are processed from primary miRNA transcripts (pri-miRNAs), many of which are annotated as long noncoding RNAs (lncRNAs). We assessed whether MIR205HG, the host gene for miR-205, has independent functions as an lncRNA. Comparing mice with targeted deletions of MIR205HG and miR-205 revealed a functional role for the lncRNA in the anterior pituitary. Mice lacking MIR205HG had a temporal reduction in Pit1, growth hormone, and prolactin. This was mediated, in part, through the ability of this lncRNA to bind and regulate the transcriptional activity of Pit1 in conjunction with Zbtb20. Knockdown of MIR205HG in lactotropes decreased the expression of Pit1, Zbtb20, prolactin, and growth hormone, while its overexpression enhanced the levels of these transcripts. The effects of MIR205HG on the pituitary were independent of miR-205. The data support a role for MIR205HG as an lncRNA that regulates growth hormone and prolactin production in the anterior pituitary.
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Hormona del Crecimiento/biosíntesis , MicroARNs/metabolismo , Adenohipófisis/metabolismo , Prolactina/biosíntesis , ARN Largo no Codificante/metabolismo , Animales , Hormona del Crecimiento/genética , Hormona del Crecimiento/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Prolactina/genética , Prolactina/metabolismo , ARN Largo no Codificante/genética , Ratas , Factor de Transcripción Pit-1/genética , Factor de Transcripción Pit-1/metabolismo , TranscriptomaRESUMEN
Proper control of microRNA (miRNA) expression is critical for normal development and physiology, while abnormal miRNA expression is a common feature of many diseases. Dissecting mechanisms of miRNA regulation, however, is complicated by the generally poor annotation of miRNA primary transcripts (pri-miRNAs). Although some miRNAs are processed from well-defined protein coding genes, the majority of pri-miRNAs are poorly characterized noncoding RNAs, with incomplete annotation of promoters, splice sites, and polyadenylation signals. Due to the efficiency of DROSHA processing, the abundance of pri-miRNAs is very low at steady state, thereby complicating the elucidation of pri-miRNA structures. Here we describe a strategy to enrich intact pri-miRNAs and improve their coverage in RNA sequencing (RNA-seq) experiments. In addition, we outline a computational approach for reconstruction of pri-miRNA structures. This pipeline begins with raw RNA-seq reads and concludes with publication-ready visualization of pri-miRNA annotations. Together, these approaches allow the user to define and explore miRNA gene structures in a cell-type or organism of interest.