Ninette Amariglio

Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq

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 N6-methyladenosine (m6A) 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 m6A modification landscape in a transcriptome-wide manner, using a novel approach, m6A-seq, based on antibody-mediated capture and massively parallel sequencing. We identify over 12,000 m6A 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 m6A 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 m6A has a fundamental role in regulation of gene expression.

Donor-Derived Brain Tumor Following Neural Stem Cell Transplantation in an Ataxia Telangiectasia Patient

Background Neural stem cells are currently being investigated as potential therapies for neurodegenerative diseases, stroke, and trauma. However, concerns have been raised over the safety of this experimental therapeutic approach, including, for example, whether there is the potential for tumors to develop from transplanted stem cells. Methods and Findings A boy with ataxia telangiectasia (AT) was treated with intracerebellar and intrathecal injection of human fetal neural stem cells. Four years after the first treatment he was diagnosed with a multifocal brain tumor. The biopsied tumor was diagnosed as a glioneuronal neoplasm. We compared the tumor cells and the patients peripheral blood cells by fluorescent in situ hybridization using X and Y chromosome probes, by PCR for the amelogenin gene X- and Y-specific alleles, by MassArray for the ATM patient specific mutation and for several SNPs, by PCR for polymorphic microsatellites, and by human leukocyte antigen (HLA) typing. Molecular and cytogenetic studies showed that the tumor was of nonhost origin suggesting it was derived from the transplanted neural stem cells. Microsatellite and HLA analysis demonstrated that the tumor is derived from at least two donors. Conclusions This is the first report of a human brain tumor complicating neural stem cell therapy. The findings here suggest that neuronal stem/progenitor cells may be involved in gliomagenesis and provide the first example of a donor-derived brain tumor. Further work is urgently needed to assess the safety of these therapies.

A module of negative feedback regulators defines growth factor signaling.

Signaling pathways invoke interplays between forward signaling and feedback to drive robust cellular response. In this study, we address the dynamics of growth factor signaling through profiling of protein phosphorylation and gene expression, demonstrating the presence of a kinetically defined cluster of delayed early genes that function to attenuate the early events of growth factor signaling. Using epidermal growth factor receptor signaling as the major model system and concentrating on regulation of transcription and mRNA stability, we demonstrate that a number of genes within the delayed early gene cluster function as feedback regulators of immediate early genes. Consistent with their role in negative regulation of cell signaling, genes within this cluster are downregulated in diverse tumor types, in correlation with clinical outcome. More generally, our study proposes a mechanistic description of the cellular response to growth factors by defining architectural motifs that underlie the function of signaling networks.

m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation

mRNA modification regulates pluripotency When stem cells progress from an embryonic pluripotent state toward a particular lineage, molecular switches dismantle the transcription factor network that keeps the cell pluripotent. Geula et al. now show that N6-methyladenosine (m6A), a messenger RNA (mRNA) modification present on transcripts of pluripotency factors, drives this transition. Methylation destabilized mRNA transcripts and limited their translation efficiency, which promoted the timely decay of naïve pluripotency. This m6A methylation was also critical for mammalian development. Science, this issue p. 1002 A messenger RNA epigenetic modification regulates stem cell progression from the pluripotent to the differentiated state. 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 N6-methyladenosine (m6A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m6A 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. m6A 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.

DNA microarrays identification of primary and secondary target genes regulated by p53

The transcriptional program regulated by the tumor suppressor p53 was analysed using oligonucleotide microarrays. A human lung cancer cell line that expresses the temperature sensitive murine p53 was utilized to quantitate mRNA levels of various genes at different time points after shifting the temperature to 32°C. Inhibition of protein synthesis by cycloheximide (CHX) was used to distinguish between primary and secondary target genes regulated by p53. In the absence of CHX, 259 and 125 genes were up or down-regulated respectively; only 38 and 24 of these genes were up and down-regulated by p53 also in the presence of CHX and are considered primary targets in this cell line. Cluster analysis of these data using the super paramagnetic clustering (SPC) algorithm demonstrate that the primary genes can be distinguished as a single cluster among a large pool of p53 regulated genes. This procedure identified additional genes that co-cluster with the primary targets and can also be classified as such genes. In addition to cell cycle (e.g. p21, TGF-β, Cyclin E) and apoptosis (e.g. Fas, Bak, IAP) related genes, the primary targets of p53 include genes involved in many aspects of cell function, including cell adhesion (e.g. Thymosin, Smoothelin), signaling (e.g. H-Ras, Diacylglycerol kinase), transcription (e.g. ATF3, LISCH7), neuronal growth (e.g. Ninjurin, NSCL2) and DNA repair (e.g. BTG2, DDB2). The results suggest that p53 activates concerted opposing signals and exerts its effect through a diverse network of transcriptional changes that collectively alter the cell phenotype in response to stress.

Association between Common Toll-Like Receptor 4 Mutations and Severe Respiratory Syncytial Virus Disease

BACKGROUND The clinical spectrum of respiratory syncytial virus (RSV) bronchiolitis in previously healthy infants is extremely variable. Thus, it is likely that factors such as genetic heterogeneity contribute to disease severity. Toll-like receptor 4 (TLR4) and CD14 are part of a receptor complex involved in the innate immune response to RSV. METHODS The association of the TLR4 mutations (Asp299Gly and Thr399Ile) and the CD14/-159 polymorphism were analyzed in 99 infants hospitalized with severe RSV bronchiolitis (group I). Eighty-two ambulatory infants with mild RSV bronchiolitis (group II) and 90 healthy adults (group III) composed the 2 control groups. The TLR4 mutations and the CD14/-159 polymorphism were genotyped by use of reverse-transcriptase polymerase chain reaction and restriction fragment-length polymorphism analysis, respectively. RESULTS Each of the TLR4 mutations, either alone or in cosegregation, were associated with severe RSV bronchiolitis: the Asp299Gly and Thr399Ile mutations were significantly overrepresented in group I, compared with groups II and III. No association between the CD14/-159 polymorphism and RSV bronchiolitis was found. CONCLUSIONS These findings suggest that TLR4 mutations, but not the CD14/-159 polymorphism, are associated with an increased risk of severe RSV bronchiolitis in previously healthy infants.

Searching for evidence of disease and malignant cell contamination in ovarian tissue stored from hematologic cancer patients

BACKGROUND Storing ovarian tissue for fertility preservation in cancer patients carries the risk of the presence of malignant cells that could lead to recurrence of cancer after reimplantation. Methods to exclude presence of cancer cells were used to improve the safety of cryopreservation-reimplantation procedures. METHODS Fifty-eight patients with hematological malignancies were referred for the storage of ovarian tissue for fertility preservation. Investigation included preoperative imaging and histological evaluation of fresh ovarian tissue. After thawing markers to detect minimal residual disease (MRD) were used and compared with patients disease used as positive control (five patients). RESULTS Preoperative imaging detected disease in the ovaries (two patients). Conventional histology post-tissue harvesting did not disclose malignant cells (56 patients). MRD results post-thawing were negative in Hodgkins disease (CD30 immunohistochemical staining), in T- and B-cell lymphoma (PCR for T-cell receptor and Ig clones, respectively) and in two chronic myelogenous leukemia patients (RT-PCR for BCR-ABL gene expression). However, highly sensitive real-time RT-PCR was positive in one CML patient and, this alarming result avoided tissue transplantation. CONCLUSIONS Preoperative imaging prevented operations and storage of tissue with cancer. Evaluation of stored ovarian tissue for MRD using sensitive markers is essential to increase safety and to prevent reimplantation of tissue with malignant cells.

LRIG1 restricts growth factor signaling by enhancing receptor ubiquitylation and degradation

Kekkon proteins negatively regulate the epidermal growth factor receptor (EGFR) during oogenesis in Drosophila. Their structural relative in mammals, LRIG1, is a transmembrane protein whose inactivation in rodents promotes skin hyperplasia, suggesting involvement in EGFR regulation. We report upregulation of LRIG1 transcript and protein upon EGF stimulation, and physical association of the encoded protein with the four EGFR orthologs of mammals. Upregulation of LRIG1 is followed by enhanced ubiquitylation and degradation of EGFR. The underlying mechanism involves recruitment of c‐Cbl, an E3 ubiquitin ligase that simultaneously ubiquitylates EGFR and LRIG1 and sorts them for degradation. We conclude that LRIG1 evolved in mammals as a feedback negative attenuator of signaling by receptor tyrosine kinases.

The dynamic N 1 -methyladenosine methylome in eukaryotic messenger RNA

Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N6-methyladenosine (m6A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N1-methyladenosine (m1A), 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 m1A 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 m1A in promoting translation of methylated mRNA.

MIR-451 and Imatinib mesylate inhibit tumor growth of Glioblastoma stem cells

We examined the microRNA profiles of Glioblastoma stem (CD133+) and non-stem (CD133-) cell populations and found up-regulation of several miRs in the CD133- cells, including miR-451, miR-486, and miR-425, some of which may be involved in regulation of brain differentiation. Transfection of GBM cells with the above miRs inhibited neurosphere formation and transfection with the mature miR-451 dispersed neurospheres, and inhibited GBM cell growth. Furthermore, transfection of miR-451 combined with Imatinib mesylate treatment had a cooperative effect in dispersal of GBM neurospheres. In addition, we identified a target site for SMAD in the promoter region of miR-451 and showed that SMAD3 and 4 activate such a promoter-luciferase construct. Transfection of SMAD in GBM cells inhibited their growth, suggesting that SMAD may drive GBM stem cells to differentiate to CD133- cells through up-regulation of miR-451 and reduces their tumorigenicity. Identification of additional miRs and target genes that regulate GBM stem cells may provide new potential drugs for therapy.