RESUMEN
In the last decade, the notion that mRNA modifications are involved in regulation of gene expression was demonstrated in thousands of studies. To date, new technologies and methods allow accurate identification, transcriptome-wide mapping, and functional characterization of a growing number of RNA modifications, providing important insights into the biology of these marks. Most of the methods and approaches were developed for studying m6A, the most prevalent internal mRNA modification. However, unique properties of other RNA modifications stimulated the development of additional approaches. In this technical primer, we will discuss the available tools and approaches for detecting and studying different RNA modifications.
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Procesamiento Postranscripcional del ARN , ARN , Epigénesis Genética , ARN/genética , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , TranscriptomaRESUMEN
The field of epitranscriptomics examines the recently deciphered form of gene expression regulation that is mediated by type- and site-specific RNA modifications. Similarly to the role played by epigenetic mechanisms - which operate via DNA and histone modifications - epitranscriptomic modifications are involved in the control of the delicate gene expression patterns that are needed for the development and activity of the nervous system and are essential for basic and higher brain functions. Here we describe the mechanisms that are involved in the writing, erasing and reading of N6-methyladenosine, the most prevalent internal mRNA modification, and the emerging roles played by N6-methyladenosine in the nervous system.
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Encéfalo/embriología , Epigénesis Genética/fisiología , Regulación de la Expresión Génica , Transcriptoma , Adenosina/análogos & derivados , Adenosina/fisiología , Animales , Orientación del Axón , Humanos , Neurogénesis , Neuroglía/fisiología , ARN Mensajero/fisiologíaRESUMEN
Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N(6)-methyladenosine (m(6)A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N(1)-methyladenosine (m(1)A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m(1)A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m(1)A in promoting translation of methylated mRNA.
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Adenosina/análogos & derivados , ARN Mensajero/metabolismo , Regiones no Traducidas 5'/genética , Adenosina/metabolismo , Animales , Secuencia de Bases , Línea Celular , Línea Celular Tumoral , Codón Iniciador/genética , Secuencia Conservada , Epigénesis Genética , Evolución Molecular , Secuencia Rica en GC/genética , Humanos , Metilación , Ratones , Especificidad de Órganos , Iniciación de la Cadena Peptídica Traduccional/genética , Sitios de Empalme de ARN/genética , ARN Mensajero/genética , Saccharomyces cerevisiae , Transcriptoma/genéticaRESUMEN
The ribose of RNA nucleotides can be 2'-O-methylated (Nm). Despite advances in high-throughput detection, the inert chemical nature of Nm still limits sensitivity and precludes mapping in mRNA. We leveraged the differential reactivity of 2'-O-methylated and 2'-hydroxylated nucleosides to periodate oxidation to develop Nm-seq, a sensitive method for transcriptome-wide mapping of Nm with base precision. Nm-seq uncovered thousands of Nm sites in human mRNA with features suggesting functional roles.
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ARN Mensajero/genética , Secuencia de Bases , Células HeLa , Humanos , Metagenómica , Metilación , Conformación de Ácido Nucleico , ARN Mensajero/química , ARN Ribosómico/química , ARN Ribosómico/genética , Ribosa/química , TranscriptomaAsunto(s)
Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/deficiencia , Colágeno Tipo I/biosíntesis , Metilación de ADN , Enfermedad de Hodgkin/enzimología , Proteínas de Neoplasias/metabolismo , Interferencia de ARN , Procesamiento Postranscripcional del ARN , ARN Neoplásico/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Secuencia de Bases , Línea Celular Tumoral , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Islas de CpG/genética , Metilación de ADN/efectos de los fármacos , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Conjuntos de Datos como Asunto , Decitabina/farmacología , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Linfocitos/metabolismo , Metilación/efectos de los fármacos , Proteínas de Neoplasias/genética , Regiones Promotoras Genéticas/genética , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Alineación de Secuencia , ARNt Metiltransferasas/metabolismoRESUMEN
An extensive repertoire of modifications is known to underlie the versatile coding, structural and catalytic functions of RNA, but it remains largely uncharted territory. Although biochemical studies indicate that N(6)-methyladenosine (m(6)A) is the most prevalent internal modification in messenger RNA, an in-depth study of its distribution and functions has been impeded by a lack of robust analytical methods. Here we present the human and mouse m(6)A modification landscape in a transcriptome-wide manner, using a novel approach, m(6)A-seq, based on antibody-mediated capture and massively parallel sequencing. We identify over 12,000 m(6)A sites characterized by a typical consensus in the transcripts of more than 7,000 human genes. Sites preferentially appear in two distinct landmarks--around stop codons and within long internal exons--and are highly conserved between human and mouse. Although most sites are well preserved across normal and cancerous tissues and in response to various stimuli, a subset of stimulus-dependent, dynamically modulated sites is identified. Silencing the m(6)A methyltransferase significantly affects gene expression and alternative splicing patterns, resulting in modulation of the p53 (also known as TP53) signalling pathway and apoptosis. Our findings therefore suggest that RNA decoration by m(6)A has a fundamental role in regulation of gene expression.
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Adenosina/análogos & derivados , Adenosina/genética , Metaboloma , ARN/metabolismo , Empalme Alternativo , Animales , Secuencia de Bases , Línea Celular Tumoral , Secuencia Conservada , Evolución Molecular , Células Hep G2 , Humanos , Metaboloma/genética , Metilación , Metiltransferasas/deficiencia , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones , ARN/genética , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/metabolismo , Transcriptoma/genéticaRESUMEN
Relapse rates in high-risk neuroblastoma remain exceedingly high. The malignant cells that are responsible for relapse have not been identified, and mechanisms of therapy resistance remain poorly understood. Here, we used single nucleus RNA sequencing and bulk whole genome sequencing to identify and characterize the residual malignant persister cells that survive chemotherapy from a cohort of 20 matched diagnosis and definitive surgery tumor samples from patients treated with high-risk neuroblastoma induction chemotherapy. We show that persister cells share common mechanisms of chemotherapy escape including suppression of MYCN activity and activation of NF-κB signaling, the latter is further enhanced by cell-cell communication between the malignant cells and the tumor microenvironment. Overall, our work dissects the transcriptional landscape of cellular persistence in high-risk neuroblastoma and paves the way to the development of new therapeutic strategies to prevent disease relapse.
RESUMEN
The role of epigenetics in tumor onset and progression has been extensively addressed. Discoveries in the last decade completely changed our view on RNA. We now realize that its diversity lies at the base of biological complexity. Adenosine-to-inosine (A-to-I) RNA editing emerges a central generator of transcriptome diversity and regulation in higher eukaryotes. It is the posttranscriptional deamination of adenosine to inosine in double-stranded RNA catalyzed by enzymes of the adenosine deaminase acting on RNA (ADAR) family. Thought at first to be restricted to coding regions of only a few genes, recent bioinformatic analyses fueled by high-throughput sequencing revealed that it is a widespread modification affecting mostly non-coding repetitive elements in thousands of genes. The rise in scope is accompanied by discovery of a growing repertoire of functions based on differential decoding of inosine by the various cellular machineries: when recognized as guanosine, it can lead to protein recoding, alternative splicing or altered microRNA specificity; when recognized by inosine-binding proteins, it can result in nuclear retention of the transcript or its degradation. An imbalance in expression of ADAR enzymes with consequent editing dysregulation is a characteristic of human cancers. These alterations may be responsible for activating proto-oncogenes or inactivating tumor suppressors. While unlikely to be an early initiating 'hit', editing dysregulation seems to contribute to tumor progression and thus should be considered a 'driver mutation'. In this review, we examine the contribution of A-to-I RNA editing to carcinogenesis.
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Adenosina/genética , Inosina/genética , Neoplasias/genética , Edición de ARN , Adenosina/química , Animales , Humanos , Inosina/químicaRESUMEN
The prevalent m6Am mRNA cap modification was recently identified as a valid target for removal by the human obesity gene FTO along with the previously established m6A mRNA modification. However, the deposition and dynamics of m6Am in regulating obesity are unknown. Here, we investigate the liver m6A/m methylomes in mice fed on a high fat Western-diet and in ob/ob mice. We find that FTO levels are elevated in fat mice, and that genes which lost m6Am marking under obesity are overly downregulated, including the two fatty-acid-binding proteins FABP2, and FABP5. Furthermore, the cellular perturbation of FTO correspondingly affect protein levels of its targets. Notably, generally m6Am- but not m6A-methylated genes, are found to be highly enriched in metabolic processes. Finally, we deplete all m6A background via Mettl3 knockout, and unequivocally uncover the association of m6Am methylation with increased mRNA stability, translation efficiency, and higher protein expression. Together, these results strongly implicate a dynamic role for m6Am in obesity-related translation regulation.
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Adenosina/análogos & derivados , Adenosina/metabolismo , Obesidad/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Animales , Dieta Occidental , Epigenómica , Proteínas de Unión a Ácidos Grasos/metabolismo , Masculino , Metilación , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias , Estabilidad del ARN , ARN Mensajero/metabolismoRESUMEN
The cellular proteome reflects the total outcome of many regulatory mechanisms that affect the metabolism of messenger RNA (mRNA) along its pathway from synthesis to degradation. Accumulating evidence in recent years has uncovered the roles of a growing number of mRNA modifications in every step along this pathway, shaping translational output. mRNA modifications affect the translation machinery directly, by influencing translation initiation, elongation and termination, or by altering mRNA levels and subcellular localization. Features of modification-related translational control are described, charting a new and complex layer of translational regulation.
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Epigenómica , Regulación de la Expresión Génica , Transcripción Genética , Transcriptoma , Adenosina/análogos & derivados , Adenosina/química , Animales , Citidina/análogos & derivados , Citidina/química , Homeostasis , Humanos , Metilación , Extensión de la Cadena Peptídica de Translación , Iniciación de la Cadena Peptídica Traduccional , Biosíntesis de Proteínas , Seudouridina/química , ARN Mensajero/química , ARN de Transferencia/químicaRESUMEN
Proteins of the nuclear envelope have been implicated as participating in gene silencing. BAF, a DNA- and LEM domain-binding protein, has been suggested to link chromatin to the nuclear envelope. We have previously shown that LAP2beta, a LEM-domain inner nuclear membrane protein, represses transcription through binding to HDAC3 and induction of histone H4 deacetylation. We now show that LAP2zeta, the smallest LAP2 family member, is also involved in regulation of transcription. We show that similar to other LEM-domain proteins LAP2zeta interacts with BAF. LAP2zeta-YFP and BAF co-localize in the cytoplasm, and overexpression of LAP2zeta leads to reduction of nucleoplasmic BAF. Mutations in the LAP2zeta-YFP LEM domain decrease its interaction with BAF retaining the nucleo-cytoplasmic distribution of BAF. Co-expression of LAP2beta and LAP2zeta results in inhibition of LAP2beta-induced gene silencing while overexpression of LAP2zeta alone leads to a small increase in transcriptional activity of various transcription factors. Our results suggest that LAP2zeta is a transcriptional regulator acting predominantly to inhibit LAP2beta-mediated repression. LAP2zeta may function by decreasing availability of BAF. These findings could have implications in the study of nuclear lamina-associated diseases and BAF-dependent retroviral integration.
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Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Clonación Molecular , Citoplasma/metabolismo , Proteínas de Unión al ADN/genética , Histona Desacetilasas/metabolismo , Humanos , Proteínas de la Membrana/genética , Ratones , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alineación de Secuencia , Activación TranscripcionalRESUMEN
Neural progenitor cells undergo somatic retrotransposition events, mainly involving L1 elements, which can be potentially deleterious. Here, we analyze the whole genomes of 20 brain samples and 80 non-brain samples, and characterized the retrotransposition landscape of patients affected by a variety of neurodevelopmental disorders including Rett syndrome, tuberous sclerosis, ataxia-telangiectasia and autism. We report that the number of retrotranspositions in brain tissues is higher than that observed in non-brain samples and even higher in pathologic vs normal brains. The majority of somatic brain retrotransposons integrate into pre-existing repetitive elements, preferentially A/T rich L1 sequences, resulting in nested insertions. Our findings document the fingerprints of encoded endonuclease independent mechanisms in the majority of L1 brain insertion events. The insertions are "non-classical" in that they are truncated at both ends, integrate in the same orientation as the host element, and their target sequences are enriched with a CCATT motif in contrast to the classical endonuclease motif of most other retrotranspositions. We show that L1Hs elements integrate preferentially into genes associated with neural functions and diseases. We propose that pre-existing retrotransposons act as "lightning rods" for novel insertions, which may give fine modulation of gene expression while safeguarding from deleterious events. Overwhelmingly uncontrolled retrotransposition may breach this safeguard mechanism and increase the risk of harmful mutagenesis in neurodevelopmental disorders.
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Encéfalo/fisiopatología , Elementos de Nucleótido Esparcido Largo/genética , Trastornos del Neurodesarrollo/genética , Nucleótidos de Adenina/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN , Bases de Datos Genéticas , Endonucleasas/genética , Exones , Regulación de la Expresión Génica , Genes/genética , Genómica/métodos , Humanos , MicroARNs/genética , Mutación , Neuronas/metabolismo , Estadísticas no Paramétricas , Nucleótidos de Timina/genética , Secuenciación Completa del GenomaRESUMEN
Nucleus-vacuole (NV) junctions in Saccharomyces cerevisiae are formed through specific interactions between Vac8p on the vacuole membrane and Nvj1p in the nuclear envelope. Herein, we report that NV junctions in yeast promote piecemeal microautophagy of the nucleus (PMN). During PMN, teardrop-like blebs are pinched from the nucleus, released into the vacuole lumen, and degraded by soluble hydrolases. PMN occurs in rapidly dividing cells but is induced to higher levels by carbon and nitrogen starvation and is under the control of the Tor kinase nutrient-sensing pathway. Confocal and biochemical assays demonstrate that Nvj1p is degraded in a PMN-dependent manner. PMN occurs normally in apg7-delta cells and is, therefore, not dependent on macroautophagy. Transmission electron microscopy reveals that portions of the granular nucleolus are often sequestered into PMN structures. These results introduce a novel mode of selective microautophagy that targets nonessential components of the yeast nucleus for degradation and recycling in the vacuole.
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Núcleo Celular/metabolismo , Saccharomyces cerevisiae/citología , Proteína 7 Relacionada con la Autofagia , Hidrolasas/metabolismo , Lipoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Membrana Nuclear/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte VesicularRESUMEN
OBJECTIVE: Metabolic dysfunctions, such as fatty liver, obesity and insulin resistance, are among the most common contemporary diseases worldwide, and their prevalence is continuously rising. Mimp/Mtch2 is a mitochondrial carrier protein homologue, which localizes to the mitochondria and induces mitochondrial depolarization. Mimp/Mtch2 single-nucleotide polymorphism is associated with obesity in humans and its loss in mice muscle protects from obesity. Our aim was to study the effects of Mimp/Mtch2 overexpression in vivo. METHODS: Transgenic mice overexpressing Mimp/Mtch2-GFP were characterized and monitored for lipid accumulation, weight and blood glucose levels. Transgenic mice liver and kidneys were used for gene expression analysis. RESULTS: Mimp/Mtch2-GFP transgenic mice express high levels of fatty acid synthase and of ß-oxidation genes and develop fatty livers and kidneys. Moreover, high-fat diet-fed Mimp/Mtch2 mice exhibit high blood glucose levels. Our results also show that Mimp/Mtch2 is involved in lipid accumulation and uptake in cells and perhaps in human obesity. CONCLUSIONS: Mimp/Mtch2 alters lipid metabolism and may play a role in the onset of obesity and development of insulin resistance.
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Glucemia/metabolismo , Perfilación de la Expresión Génica/métodos , Riñón/patología , Hígado/patología , Proteínas de Transporte de Membrana Mitocondrial/genética , Obesidad/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Animales , Dieta Alta en Grasa , Ácido Graso Sintasas/genética , Regulación de la Expresión Génica , Riñón/metabolismo , Metabolismo de los Lípidos , Hígado/metabolismo , Potencial de la Membrana Mitocondrial , Ratones , Ratones Transgénicos , Proteínas de Transporte de Membrana Mitocondrial/metabolismoRESUMEN
Polymorphism of the FTO gene encoding an N(6)-methyladenosine (m(6)A) RNA demethylase was robustly associated with human obesity; however, the mechanism by which FTO affects metabolism, considering its emerging role in RNA modification, is still poorly understood. A new study published in Cell Research reports novel functions implicating FTO in the regulation of mRNA alternative splicing in the control of adipogenesis.
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Adenosina/análogos & derivados , Adipocitos/citología , Adipogénesis , Oxigenasas de Función Mixta/metabolismo , Oxo-Ácido-Liasas/metabolismo , Empalme del ARN , ARN Mensajero/genética , AnimalesRESUMEN
A detailed protocol for isolation and sequencing of an enriched population of m(6)A-methylated RNA fragments to create m(6)A methylome maps is outlined. Our approach was developed to fill a void that existed because of a lack of methods for the detection of m(6)A in RNA in an unbiased, high-throughput, and high-resolution manner. This method integrates immunoprecipitation of methylated, randomly fragmented RNA using a highly specific anti-m(6)A antibody to obtain an enriched population of modified fragments and massively parallel sequencing, resulting in mapping of this modification throughout the transcriptome.
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Adenosina/análogos & derivados , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN/química , Transcriptoma/genética , Adenosina/química , Adenosina/genética , Adenosina/aislamiento & purificación , Metilación , ARN/genéticaRESUMEN
Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N(6)-methyladenosine (m(6)A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m(6)A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m(6)A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner.
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Adenosina/análogos & derivados , Diferenciación Celular/fisiología , Metiltransferasas/fisiología , Células Madre Pluripotentes/citología , ARN Mensajero/metabolismo , Adenosina/metabolismo , Animales , Blastocisto/enzimología , Diferenciación Celular/genética , Línea Celular , Pérdida del Embrión/genética , Epigénesis Genética , Femenino , Técnicas de Inactivación de Genes , Masculino , Metilación , Metiltransferasas/genética , Ratones , Ratones Noqueados , Células Madre Pluripotentes/enzimologíaRESUMEN
N(6)-methyladenosine-sequencing (m(6)A-seq) is an immunocapturing approach for the unbiased transcriptome-wide localization of m(6)A in high resolution. To our knowledge, this is the first protocol to allow a global view of this ubiquitous RNA modification, and it is based on antibody-mediated enrichment of methylated RNA fragments followed by massively parallel sequencing. Building on principles of chromatin immunoprecipitation-sequencing (ChIP-seq) and methylated DNA immunoprecipitation (MeDIP), read densities of immunoprecipitated RNA relative to untreated input control are used to identify methylated sites. A consensus motif is deduced, and its distance to the point of maximal enrichment is assessed; these measures further corroborate the success of the protocol. Identified locations are intersected in turn with gene architecture to draw conclusions regarding the distribution of m(6)A between and within gene transcripts. When applied to human and mouse transcriptomes, m(6)A-seq generated comprehensive methylation profiles revealing, for the first time, tenets governing the nonrandom distribution of m(6)A. The protocol can be completed within ~9 d for four different sample pairs (each consists of an immunoprecipitation and corresponding input).
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Adenosina/análogos & derivados , Adenosina/química , Perfilación de la Expresión Génica/métodos , Inmunoprecipitación/métodos , Procesamiento Postranscripcional del ARN , Animales , Humanos , Metilación , RatonesRESUMEN
Molecular functional and metabolic imaging allows visualization of disease-causing processes in living organisms. Here we present a new approach for the functional molecular imaging (FMI) of endogenous tyrosine kinase receptor activity using Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), as a model. HGF/SF and Met play significant roles in the biology and pathogenesis of a wide variety of cancers and, therefore, may serve as potential targets for cancer prognosis and therapy. We have previously shown that Met activation by HGF/SF increases oxygen consumption in vitro and results in substantial alteration of blood oxygenation levels in vivo, as measured by blood oxygenation level-dependent magnetic resonance imaging. Using contrast medium (CM) ultrasound imaging, we demonstrate here that HGF/SF induces an increase in tumor blood volume. This increase is evident in small vessels, including vessels that were not detected before HGF/SF treatment. The specificity of the effect was validated by its inhibition using anti-HGF/SF antibodies. This change in tumor hemodynamics, induced by HGF/SF and measured by CM ultrasound, is further used as a tool for Met FMI in tumors. This novel noninvasive molecular imaging technique may be applied for the in vivo diagnosis, prognosis, and therapy of Met-expressing tumors.