RESUMEN
mTORC1 is a signal integrator and master regulator of cellular anabolic processes linked to cell growth and survival. Here, we demonstrate that mTORC1 promotes lipid biogenesis via SRPK2, a key regulator of RNA-binding SR proteins. mTORC1-activated S6K1 phosphorylates SRPK2 at Ser494, which primes Ser497 phosphorylation by CK1. These phosphorylation events promote SRPK2 nuclear translocation and phosphorylation of SR proteins. Genome-wide transcriptome analysis reveals that lipid biosynthetic enzymes are among the downstream targets of mTORC1-SRPK2 signaling. Mechanistically, SRPK2 promotes SR protein binding to U1-70K to induce splicing of lipogenic pre-mRNAs. Inhibition of this signaling pathway leads to intron retention of lipogenic genes, which triggers nonsense-mediated mRNA decay. Genetic or pharmacological inhibition of SRPK2 blunts de novo lipid synthesis, thereby suppressing cell growth. These results thus reveal a novel role of mTORC1-SRPK2 signaling in post-transcriptional regulation of lipid metabolism and demonstrate that SRPK2 is a potential therapeutic target for mTORC1-driven metabolic disorders.
Asunto(s)
Regulación de la Expresión Génica , Lipogénesis , Procesamiento Postranscripcional del ARN , Transducción de Señal , Animales , Núcleo Celular/metabolismo , Colesterol/metabolismo , Ácidos Grasos/metabolismo , Femenino , Xenoinjertos , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismoRESUMEN
SARS-CoV-2 RNA interacts with host factors to suppress interferon responses and simultaneously induces cytokine release to drive the development of severe coronavirus disease 2019 (COVID-19). However, how SARS-CoV-2 hijacks host RNAs to elicit such imbalanced immune responses remains elusive. Here, we analyzed SARS-CoV-2 RNA in situ structures and interactions in infected cells and patient lung samples using RIC-seq. We discovered that SARS-CoV-2 RNA forms 2,095 potential duplexes with the 3' UTRs of 205 host mRNAs to increase their stability by recruiting RNA-binding protein YBX3 in A549 cells. Disrupting the SARS-CoV-2-to-host RNA duplex or knocking down YBX3 decreased host mRNA stability and reduced viral replication. Among SARS-CoV-2-stabilized host targets, NFKBIZ was crucial for promoting cytokine production and reducing interferon responses, probably contributing to cytokine storm induction. Our study uncovers the crucial roles of RNA-RNA interactions in the immunopathogenesis of RNA viruses such as SARS-CoV-2 and provides valuable host targets for drug development.
Asunto(s)
COVID-19 , Humanos , COVID-19/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , ARN Viral/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Interferones/genética , CitocinasRESUMEN
2'-O-methylation (Nm) is a prominent RNA modification well known in noncoding RNAs and more recently also found at many mRNA internal sites. However, their function and base-resolution stoichiometry remain underexplored. Here, we investigate the transcriptome-wide effect of internal site Nm on mRNA stability. Combining nanopore sequencing with our developed machine learning method, NanoNm, we identify thousands of Nm sites on mRNAs with a single-base resolution. We observe a positive effect of FBL-mediated Nm modification on mRNA stability and expression level. Elevated FBL expression in cancer cells is associated with increased expression levels for 2'-O-methylated mRNAs of cancer pathways, implying the role of FBL in post-transcriptional regulation. Lastly, we find that FBL-mediated 2'-O-methylation connects to widespread 3' UTR shortening, a mechanism that globally increases RNA stability. Collectively, we demonstrate that FBL-mediated Nm modifications at mRNA internal sites regulate gene expression by enhancing mRNA stability.
Asunto(s)
Regiones no Traducidas 3' , Estabilidad del ARN , ARN Mensajero , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Metilación , Procesamiento Postranscripcional del ARN , Secuenciación de Nanoporos/métodos , Transcriptoma , Regulación Neoplásica de la Expresión Génica , Aprendizaje AutomáticoRESUMEN
Mitochondria are essential regulators of innate immunity. They generate long mitochondrial double-stranded RNAs (mt-dsRNAs) and release them into the cytosol to trigger an immune response under pathological stress conditions. Yet the regulation of these self-immunogenic RNAs remains largely unknown. Here, we employ CRISPR screening on mitochondrial RNA (mtRNA)-binding proteins and identify NOP2/Sun RNA methyltransferase 4 (NSUN4) as a key regulator of mt-dsRNA expression in human cells. We find that NSUN4 induces 5-methylcytosine (m5C) modification on mtRNAs, especially on the termini of light-strand long noncoding RNAs. These m5C-modified RNAs are recognized by complement C1q-binding protein (C1QBP), which recruits polyribonucleotide nucleotidyltransferase to facilitate RNA turnover. Suppression of NSUN4 or C1QBP results in increased mt-dsRNA expression, while C1QBP deficiency also leads to increased cytosolic mt-dsRNAs and subsequent immune activation. Collectively, our study unveils the mechanism underlying the selective degradation of light-strand mtRNAs and establishes a molecular mark for mtRNA decay and cytosolic release.
Asunto(s)
5-Metilcitosina , Citosol , Mitocondrias , Estabilidad del ARN , ARN Bicatenario , ARN Mitocondrial , Humanos , Citosol/metabolismo , 5-Metilcitosina/metabolismo , Mitocondrias/metabolismo , Mitocondrias/genética , ARN Bicatenario/metabolismo , ARN Bicatenario/genética , ARN Mitocondrial/genética , ARN Mitocondrial/metabolismo , Células HEK293 , Células HeLa , Metiltransferasas/metabolismo , Metiltransferasas/genética , Inmunidad Innata , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Animales , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Sistemas CRISPR-CasRESUMEN
The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth that stimulates macromolecule synthesis through transcription, RNA processing, and post-translational modification of metabolic enzymes. However, the mechanisms of how mTORC1 orchestrates multiple steps of gene expression programs remain unclear. Here, we identify family with sequence similarity 120A (FAM120A) as a transcription co-activator that couples transcription and splicing of de novo lipid synthesis enzymes downstream of mTORC1-serine/arginine-rich protein kinase 2 (SRPK2) signaling. The mTORC1-activated SRPK2 phosphorylates splicing factor serine/arginine-rich splicing factor 1 (SRSF1), enhancing its binding to FAM120A. FAM120A directly interacts with a lipogenic transcription factor SREBP1 at active promoters, thereby bridging the newly transcribed lipogenic genes from RNA polymerase II to the SRSF1 and U1-70K-containing RNA-splicing machinery. This mTORC1-regulated, multi-protein complex promotes efficient splicing and stability of lipogenic transcripts, resulting in fatty acid synthesis and cancer cell proliferation. These results elucidate FAM120A as a critical transcription co-factor that connects mTORC1-dependent gene regulation programs for anabolic cell growth.
Asunto(s)
Arginina , Lipogénesis , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Lipogénesis/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Factores de Empalme de ARN , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Humanos , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
Information in mRNA has largely been thought to be confined to its nucleotide sequence. However, the advent of mapping techniques to detect modified nucleotides has revealed that mRNA contains additional information in the form of chemical modifications. The most abundant modified nucleotide is N6-methyladenosine (m6A), a methyl modification of adenosine. Although early studies viewed m6A as a dynamic and tissue-specific modification, it is now clear that the mRNAs that contain m6A and the location of m6A in those transcripts are largely universal and are influenced by gene architecture, i.e., the size and location of exons and introns. m6A can affect nuclear processes such as splicing and epigenetic regulation, but the major effect of m6A on mRNAs is to promote degradation in the cytoplasm. m6A marks a functionally related cohort of mRNAs linked to certain biological processes, including cell differentiation and cell fate determination. m6A is also enriched in other cohorts of mRNAs and can therefore affect their respective cellular processes and pathways. Future work will focus on understanding how the m6A pathway is regulated to achieve control of m6A-containing mRNAs.
Asunto(s)
Adenosina , Epigénesis Genética , Adenosina/genética , Adenosina/metabolismo , Expresión Génica , Humanos , Metiltransferasas/genética , Metiltransferasas/metabolismo , Nucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
R-2-hydroxyglutarate (R-2HG), a metabolite produced by mutant isocitrate dehydrogenases (IDHs), was recently reported to exhibit anti-tumor activity. However, its effect on cancer metabolism remains largely elusive. Here we show that R-2HG effectively attenuates aerobic glycolysis, a hallmark of cancer metabolism, in (R-2HG-sensitive) leukemia cells. Mechanistically, R-2HG abrogates fat-mass- and obesity-associated protein (FTO)/N6-methyladenosine (m6A)/YTH N6-methyladenosine RNA binding protein 2 (YTHDF2)-mediated post-transcriptional upregulation of phosphofructokinase platelet (PFKP) and lactate dehydrogenase B (LDHB) (two critical glycolytic genes) expression and thereby suppresses aerobic glycolysis. Knockdown of FTO, PFKP, or LDHB recapitulates R-2HG-induced glycolytic inhibition in (R-2HG-sensitive) leukemia cells, but not in normal CD34+ hematopoietic stem/progenitor cells, and inhibits leukemogenesis in vivo; conversely, their overexpression reverses R-2HG-induced effects. R-2HG also suppresses glycolysis and downregulates FTO/PFKP/LDHB expression in human primary IDH-wild-type acute myeloid leukemia (AML) cells, demonstrating the clinical relevance. Collectively, our study reveals previously unrecognized effects of R-2HG and RNA modification on aerobic glycolysis in leukemia, highlighting the therapeutic potential of targeting cancer epitranscriptomics and metabolism.
Asunto(s)
Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Antineoplásicos/farmacología , Glutaratos/farmacología , Glucólisis/genética , Lactato Deshidrogenasas/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Fosfofructoquinasa-1 Tipo C/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/antagonistas & inhibidores , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Fluorouracilo/farmacología , Regulación Neoplásica de la Expresión Génica , Glucólisis/efectos de los fármacos , Células HEK293 , Humanos , Células K562 , Lactato Deshidrogenasas/antagonistas & inhibidores , Lactato Deshidrogenasas/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación Oxidativa/efectos de los fármacos , Fosfofructoquinasa-1 Tipo C/antagonistas & inhibidores , Fosfofructoquinasa-1 Tipo C/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Análisis de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In animals, PIWI-interacting RNAs (piRNAs) silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3' terminal trimming and 2'-O-methylation. Both trimming and methylation influence piRNA stability. Our biochemical data show that multiple mechanisms destabilize unmethylated mouse piRNAs, depending on whether the piRNA 5' or 3' sequence is complementary to a trigger RNA. Unlike target-directed degradation of microRNAs, complementarity-dependent destabilization of piRNAs in mice and flies is blocked by 3' terminal 2'-O-methylation and does not require base pairing to both the piRNA seed and the 3' sequence. In flies, 2'-O-methylation also protects small interfering RNAs (siRNAs) from complementarity-dependent destruction. By contrast, pre-piRNA trimming protects mouse piRNAs from a degradation pathway unaffected by trigger complementarity. In testis lysate and in vivo, internal or 3' terminal uridine- or guanine-rich tracts accelerate pre-piRNA decay. Loss of both trimming and 2'-O-methylation causes the mouse piRNA pathway to collapse, demonstrating that these modifications collaborate to stabilize piRNAs.
Asunto(s)
Proteínas Argonautas/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Separación Celular , Drosophila melanogaster , Femenino , Citometría de Flujo , Expresión Génica , Silenciador del Gen , Técnicas Genéticas , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Procesamiento Proteico-Postraduccional , ARN Bicatenario , Espermatocitos/metabolismo , Espermatogonias/metabolismo , Testículo/metabolismoRESUMEN
RNase E is an essential, multifunctional ribonuclease encoded in E. coli by the rne gene. Structural analysis indicates that the ribonucleolytic activity of this enzyme is conferred by rne-encoded polypeptide chains that (1) dimerize to form a catalytic site at the protein-protein interface, and (2) multimerize further to generate a tetrameric quaternary structure consisting of two dimerized Rne-peptide chains. We identify here a mutation in the Rne protein's catalytic region (E429G), as well as a bacterial cell wall peptidoglycan hydrolase (Amidase C [AmiC]), that selectively affect the specific activity of the RNase E enzyme on long RNA substrates, but not on short synthetic oligonucleotides, by enhancing enzyme multimerization. Unlike the increase in specific activity that accompanies concentration-induced multimerization, enhanced multimerization associated with either the E429G mutation or interaction of the Rne protein with AmiC is independent of the substrate's 5' terminus phosphorylation state. Our findings reveal a previously unsuspected substrate length-dependent regulatory role for RNase E quaternary structure and identify cis-acting and trans-acting factors that mediate such regulation.
Asunto(s)
Endorribonucleasas/química , Endorribonucleasas/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Escherichia coli/genética , Modelos Moleculares , Amidohidrolasas/metabolismo , Dominio Catalítico , Endorribonucleasas/genética , Proteínas de Escherichia coli/genética , Mutación/genética , Estructura Cuaternaria de Proteína , ARN Bacteriano/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Previous work has demonstrated that the epitranscriptomic addition of m6A to viral transcripts can promote the replication and pathogenicity of a wide range of DNA and RNA viruses, including HIV-1, yet the underlying mechanisms responsible for this effect have remained unclear. It is known that m6A function is largely mediated by cellular m6A binding proteins or readers, yet how these regulate viral gene expression in general, and HIV-1 gene expression in particular, has been controversial. Here, we confirm that m6A addition indeed regulates HIV-1 RNA expression and demonstrate that this effect is largely mediated by the nuclear m6A reader YTHDC1 and the cytoplasmic m6A reader YTHDF2. Both YTHDC1 and YTHDF2 bind to multiple distinct and overlapping sites on the HIV-1 RNA genome, with YTHDC1 recruitment serving to regulate the alternative splicing of HIV-1 RNAs. Unexpectedly, while YTHDF2 binding to m6A residues present on cellular mRNAs resulted in their destabilization as previously reported, YTHDF2 binding to m6A sites on HIV-1 transcripts resulted in a marked increase in the stability of these viral RNAs. Thus, YTHDF2 binding can exert diametrically opposite effects on RNA stability, depending on RNA sequence context.
Asunto(s)
VIH-1 , Adenosina/metabolismo , Empalme Alternativo , VIH-1/genética , VIH-1/metabolismo , Empalme del ARN , Estabilidad del ARN/genética , ARN Viral/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
ADP-ribosylation (ADPRylation) is a posttranslational modification of proteins discovered nearly six decades ago, but many important questions remain regarding its molecular functions and biological roles, as well as the activity of the ADP-ribose (ADPR) transferase enzymes (PARP family members) that catalyze it. Growing evidence indicates that PARP-mediated ADPRylation events are key regulators of the protein biosynthetic pathway, leading from rDNA transcription and ribosome biogenesis to mRNA synthesis, processing, and translation. In this review we describe the role of PARP proteins and ADPRylation in all facets of this pathway. PARP-1 and its enzymatic activity are key regulators of rDNA transcription, which is a critical step in ribosome biogenesis. An emerging role of PARPs in alternative splicing of mRNAs, as well as direct ADPRylation of mRNAs, highlight the role of PARP members in RNA processing. Furthermore, PARP activity, stimulated by cellular stresses, such as viral infections and ER stress, leads to the regulation of mRNA stability and protein synthesis through posttranscriptional mechanisms. Dysregulation of PARP activity in these processes can promote disease states. Collectively, these results highlight the importance of PARP family members and ADPRylation in gene regulation, mRNA processing, and protein abundance. Future studies in these areas will yield new insights into the fundamental mechanisms and a broader utility for PARP-targeted therapeutic agents.
Asunto(s)
ADP-Ribosilación/fisiología , Expresión Génica/fisiología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Biosíntesis de Proteínas/fisiología , Proteostasis/fisiología , Animales , Humanos , Procesamiento Proteico-Postraduccional , ARN/metabolismoRESUMEN
Alternative pre-mRNA-splicing-induced post-transcriptional gene expression regulation is one of the pathways for tumors maintaining proliferation rates accompanying the malignant phenotype under stress. Here, we uncover a list of hyperacetylated proteins in the context of acutely reduced Acetyl-CoA levels under nutrient starvation. PHF5A, a component of U2 snRNPs, can be acetylated at lysine 29 in response to multiple cellular stresses, which is dependent on p300. PHF5A acetylation strengthens the interaction among U2 snRNPs and affects global pre-mRNA splicing pattern and extensive gene expression. PHF5A hyperacetylation-induced alternative splicing stabilizes KDM3A mRNA and promotes its protein expression. Pathologically, PHF5A K29 hyperacetylation and KDM3A upregulation axis are correlated with poor prognosis of colon cancer. Our findings uncover a mechanism of an anti-stress pathway through which acetylation on PHF5A promotes the cancer cells' capacity for stress resistance and consequently contributes to colon carcinogenesis.
Asunto(s)
Empalme Alternativo , Carcinogénesis/genética , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Histona Demetilasas con Dominio de Jumonji/genética , Proteínas de Unión al ARN/genética , Transactivadores/genética , Acetilcoenzima A/deficiencia , Acetilación , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Células HCT116 , Humanos , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/metabolismo , Células MCF-7 , Masculino , Ratones , Ratones Desnudos , Pronóstico , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/genética , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Transducción de Señal , Análisis de Supervivencia , Transactivadores/antagonistas & inhibidores , Transactivadores/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The potential presence of 5-methylcytosine as a sparse internal modification of mRNA was first raised in 1975, and a first map of the modification was also part of the epitranscriptomics "big bang" in 2012. Since then, the evidence for its presence in mRNA has firmed up, and initial insights have been gained into the molecular function and broader biological relevance of 5-methylcytosine when present in mRNA. Here, we summarize the status quo of the field, outline some of its current challenges, and suggest how to address them in future work.
Asunto(s)
5-Metilcitosina , ARN , ARN Mensajero/genéticaRESUMEN
Bacterial protein synthesis rates have evolved to maintain preferred stoichiometries at striking precision, from the components of protein complexes to constituents of entire pathways. Setting relative protein production rates to be well within a factor of two requires concerted tuning of transcription, RNA turnover, and translation, allowing many potential regulatory strategies to achieve the preferred output. The last decade has seen a greatly expanded capacity for precise interrogation of each step of the central dogma genome-wide. Here, we summarize how these technologies have shaped the current understanding of diverse bacterial regulatory architectures underpinning stoichiometric protein synthesis. We focus on the emerging expanded view of bacterial operons, which encode diverse primary and secondary mRNA structures for tuning protein stoichiometry. Emphasis is placed on how quantitative tuning is achieved. We discuss the challenges and open questions in the application of quantitative, genome-wide methodologies to the problem of precise protein production.
Asunto(s)
Escherichia coli , Operón , Escherichia coli/genética , Biosíntesis de Proteínas , Proteínas/metabolismo , ARN Mensajero/metabolismo , Transcripción GenéticaRESUMEN
Nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their stability, function, and potential use as disease biomarkers. Herein, we measured the stability of several naked RNAs when incubated in human serum, urine, and cerebrospinal fluid (CSF). We identified extracellularly produced tRNA-derived small RNAs (tDRs) with half-lives of several hours in CSF. Contrary to widespread assumptions, these intrinsically stable small RNAs are full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs). Standard molecular biology protocols, including phenol-based RNA extraction and heat, induce the artifactual denaturation of nicked tRNAs and the consequent in vitro production of tDRs. Broken bonds are roadblocks for reverse transcriptases, preventing amplification and/or sequencing of nicked tRNAs in their native state. To solve this, we performed enzymatic repair of nicked tRNAs purified under native conditions, harnessing the intrinsic activity of phage and bacterial tRNA repair systems. Enzymatic repair regenerated an RNase R-resistant tRNA-sized band in northern blot and enabled RT-PCR amplification of full-length tRNAs. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions, identifying nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Dissociation of nicked tRNAs produces single-stranded tDRs that can be spontaneously taken up by human epithelial cells, positioning stable nv-exRNAs as potentially relevant players in intercellular communication pathways.
Asunto(s)
ARN de Transferencia , ARN , Humanos , ARN de Transferencia/metabolismo , Bacterias/metabolismo , Células Epiteliales/metabolismoRESUMEN
BACKGROUND: Discovering determinants of cardiomyocyte maturity is critical for deeply understanding the maintenance of differentiated states and potentially reawakening endogenous regenerative programs in adult mammalian hearts as a therapeutic strategy. Forced dedifferentiation paired with oncogene expression is sufficient to drive cardiac regeneration, but elucidation of endogenous developmental regulators of the switch between regenerative and mature cardiomyocyte cell states is necessary for optimal design of regenerative approaches for heart disease. MBNL1 (muscleblind-like 1) regulates fibroblast, thymocyte, and erythroid differentiation and proliferation. Hence, we examined whether MBNL1 promotes and maintains mature cardiomyocyte states while antagonizing cardiomyocyte proliferation. METHODS: MBNL1 gain- and loss-of-function mouse models were studied at several developmental time points and in surgical models of heart regeneration. Multi-omics approaches were combined with biochemical, histological, and in vitro assays to determine the mechanisms through which MBNL1 exerts its effects. RESULTS: MBNL1 is coexpressed with a maturation-association genetic program in the heart and is regulated by the MEIS1/calcineurin signaling axis. Targeted MBNL1 overexpression early in development prematurely transitioned cardiomyocytes to hypertrophic growth, hypoplasia, and dysfunction, whereas loss of MBNL1 function increased cardiomyocyte cell cycle entry and proliferation through altered cell cycle inhibitor transcript stability. Moreover, MBNL1-dependent stabilization of estrogen-related receptor signaling was essential for maintaining cardiomyocyte maturity in adult myocytes. In accordance with these data, modulating MBNL1 dose tuned the temporal window of neonatal cardiac regeneration, where increased MBNL1 expression arrested myocyte proliferation and regeneration and MBNL1 deletion promoted regenerative states with prolonged myocyte proliferation. However, MBNL1 deficiency was insufficient to promote regeneration in the adult heart because of cell cycle checkpoint activation. CONCLUSIONS: Here, MBNL1 was identified as an essential regulator of cardiomyocyte differentiated states, their developmental switch from hyperplastic to hypertrophic growth, and their regenerative potential through controlling an entire maturation program by stabilizing adult myocyte mRNAs during postnatal development and throughout adulthood. Targeting loss of cardiomyocyte maturity and downregulation of cell cycle inhibitors through MBNL1 deletion was not sufficient to promote adult regeneration.
Asunto(s)
Diferenciación Celular , Miocitos Cardíacos , Proteínas de Unión al ARN , Regeneración , Animales , Miocitos Cardíacos/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ratones , Proliferación Celular , Transducción de Señal , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/metabolismo , Proteínas de Unión al ADNRESUMEN
The conserved family of RNA-binding proteins (RBPs), IGF2BPs, plays an essential role in posttranscriptional regulation controlling mRNA stability, localization, and translation. Mammalian cells express three isoforms of IGF2BPs: IGF2BP1-3. IGF2BP3 is highly overexpressed in cancer cells, and its expression correlates with a poor prognosis in various tumors. Therefore, revealing its target RNAs with high specificity in healthy tissues and in cancer cells is of crucial importance. Photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) identifies the binding sites of RBPs on their target RNAs at nucleotide resolution in a transcriptome-wide manner. Here, we optimized the PAR-CLIP protocol to study RNA targets of endogenous IGF2BP3 in a human colorectal carcinoma cell line. To this end, we first established an immunoprecipitation protocol to obtain highly pure endogenous IGF2BP3-RNA complexes. Second, we modified the protocol to use highly sensitive infrared (IR) fluorescent dyes instead of radioactive probes to visualize IGF2BP3-crosslinked RNAs. We named the modified method "IR-PAR-CLIP." Third, we compared RNase cleavage conditions and found that sequence preferences of the RNases impact the number of the identified IGF2BP3 targets and introduce a systematic bias in the identified RNA motifs. Fourth, we adapted the single adapter circular ligation approach to increase the efficiency in library preparation. The optimized IR-PAR-CLIP protocol revealed novel RNA targets of IGF2BP3 in a human colorectal carcinoma cell line. We anticipate that our IR-PAR-CLIP approach provides a framework for studies of other RBPs.
Asunto(s)
Neoplasias del Colon , Neoplasias Colorrectales , Ribonucleósidos , Animales , Humanos , ARN/genética , Inmunoprecipitación , Proteínas de Unión al ARN/metabolismo , Sitios de Unión , Ribonucleasas/metabolismo , Ribonucleósidos/química , Mamíferos/genéticaRESUMEN
In this issue of Molecular Cell, Chao et al. (2017) investigate the important role of the low-specificity endonuclease RNase E in shaping the transcriptome of a bacterial pathogen by functioning as both a degradative enzyme and an RNA maturase.
Asunto(s)
Escherichia coli/enzimología , ARN Mensajero/genética , Endorribonucleasas/genética , ARN BacterianoRESUMEN
The stability of mRNAs is regulated by signals within their sequences, but a systematic and predictive understanding of the underlying sequence rules remains elusive. Here we introduce UTR-seq, a combination of massively parallel reporter assays and regression models, to survey the dynamics of tens of thousands of 3' UTR sequences during early zebrafish embryogenesis. UTR-seq revealed two temporal degradation programs: a maternally encoded early-onset program and a late-onset program that accelerated degradation after zygotic genome activation. Three signals regulated early-onset rates: stabilizing poly-U and UUAG sequences and destabilizing GC-rich signals. Three signals explained late-onset degradation: miR-430 seeds, AU-rich sequences, and Pumilio recognition sites. Sequence-based regression models translated 3' UTRs into their unique decay patterns and predicted the in vivo effect of sequence signals on mRNA stability. Their application led to the successful design of artificial 3' UTRs that conferred specific mRNA dynamics. UTR-seq provides a general strategy to uncover the rules of RNA cis regulation.
Asunto(s)
Regiones no Traducidas 3' , Embrión no Mamífero/metabolismo , Genes Reporteros , Estabilidad del ARN , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Cigoto/metabolismo , Animales , Embrión no Mamífero/citología , Regulación de la Expresión Génica , MicroARNs , ARN Mensajero , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Cigoto/crecimiento & desarrolloRESUMEN
IGF2BP2 binds to a number of RNA transcripts and has been suggested to function as a tumor promoter, although little is known regarding the mechanisms that regulate its roles in RNA metabolism. Here we demonstrate that IGF2BP2 binds to the 3' untranslated region of the transcript encoding ATP6V1A, a catalytic subunit of the vacuolar ATPase (v-ATPase), and serves as a substrate for the NAD+-dependent deacetylase SIRT1, which regulates how IGF2BP2 affects the stability of the ATP6V1A transcript. When sufficient levels of SIRT1 are expressed, it catalyzes the deacetylation of IGF2BP2, which can bind to the ATP6V1A transcript but does not mediate its degradation. However, when SIRT1 expression is low, the acetylated form of IGF2BP2 accumulates, and upon binding to the ATP6V1A transcript recruits the XRN2 nuclease, which catalyzes transcript degradation. Thus, the stability of the ATP6V1A transcript is significantly compromised in breast cancer cells when SIRT1 expression is low or knocked-down. This leads to a reduction in the expression of functional v-ATPase complexes in cancer cells and to an impairment in their lysosomal activity, resulting in the production of a cellular secretome consisting of increased numbers of exosomes enriched in ubiquitinated protein cargo and soluble hydrolases, including cathepsins, that together combine to promote tumor cell survival and invasiveness. These findings describe a previously unrecognized role for IGF2BP2 in mediating the degradation of a messenger RNA transcript essential for lysosomal function and highlight how its sirtuin-regulated acetylation state can have significant biological and disease consequences.