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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. In this study, 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 MYC(N) activity and activation of NFκB signaling, and 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. Significance: Approximately 50% of patients with high-risk neuroblastoma die of relapsed refractory disease. We identified the malignant cells that likely contribute to relapse and discovered key signaling pathways that mediate cellular persistence. Inhibition of these pathways and their downstream effectors is postulated to eliminate persister cells and prevent relapse. See related commentary by Wolf et al., p. 2308., (©2024 The Authors; Published by the American Association for Cancer Research.)

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 m 6 A, 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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The prevalent m 6 Am mRNA cap modification was recently identified as a valid target for removal by the human obesity gene FTO along with the previously established m 6 A mRNA modification. However, the deposition and dynamics of m 6 Am in regulating obesity are unknown. Here, we investigate the liver m 6 A/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 m 6 Am 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 m 6 Am- but not m 6 A-methylated genes, are found to be highly enriched in metabolic processes. Finally, we deplete all m 6 A background via Mettl3 knockout, and unequivocally uncover the association of m 6 Am methylation with increased mRNA stability, translation efficiency, and higher protein expression. Together, these results strongly implicate a dynamic role for m 6 Am in obesity-related translation regulation., (© 2021. The Author(s).)

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 N 6 -methyladenosine, the most prevalent internal mRNA modification, and the emerging roles played by N 6 -methyladenosine in the nervous system.

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., (Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved.)

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.

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.

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 β-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.

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.

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., (Copyright © 2015, American Association for the Advancement of Science.)

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.

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., (© 2015 Elsevier Inc. All rights reserved.)

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).

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.

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.

The tyrosine kinase receptor Met and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), play an important role in normal developmental processes, as well as in tumorigenicity and metastasis. We constructed a green fluorescent protein (GFP) Met chimeric molecule that functions similarly to the wild-type Met receptor and generated GFP-Met transgenic mice. These mice ubiquitously expressed GFP-Met in specific epithelial and endothelial cells and displayed enhanced GFP-Met fluorescence in sebaceous glands. Thirty-two percent of males spontaneously developed adenomas, adenocarcinomas, and angiosarcomas in their lower abdominal sebaceous glands. Approximately 70% of adenocarcinoma tumors metastasized to the kidneys, lungs, or liver. Quantitative subcellular-resolution intravital imaging revealed very high levels of GFP-Met in tumor lesions and in single isolated cells surrounding them, relative to normal sebaceous glands. These single cells preceded the formation of local and distal metastases. Higher GFP-Met levels correlated with earlier tumor onset and aggressiveness, further demonstrating the role of Met-HGF/SF signaling in cellular transformation and acquisition of invasive and metastatic phenotypes. Our novel mouse model and high-resolution intravital molecular imaging create a powerful tool that enables direct real-time molecular imaging of receptor expression and localization during primary events of tumorigenicity and metastasis at single-cell resolution.

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.

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.