Dan Dominissini

Bio: Dan Dominissini is an academic researcher from Sheba Medical Center. The author has contributed to research in topics: Gene expression & Transcriptome. The author has an hindex of 7, co-authored 12 publications receiving 2926 citations. Previous affiliations of Dan Dominissini include Tel Aviv University & University of Chicago.

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

Abstract: 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.

Transcriptome-wide mapping of N6-methyladenosine by m6A-seq based on immunocapturing and massively parallel sequencing

Abstract: 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)

N(6)-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics

Abstract: N(6)-methylation of adenosine (forming m(6)A) is the most abundant post-transcriptional modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m(6)A in mRNA decoding. Although m(6)A base-pairs with uridine during decoding, as shown by X-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements in an Escherichia coli translation system revealed that m(6)A modification of mRNA acts as a barrier to tRNA accommodation and translation elongation. The interaction between an m(6)A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, because delay in tRNA accommodation depends on the position and context of m(6)A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics.

N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection.

Abstract: Methylation at the N6 position of adenosine (m6A) is a highly prevalent and reversible modification within eukaryotic mRNAs that has been linked to many stages of RNA processing and fate. Recent studies suggest that m6A deposition and proteins involved in the m6A pathway play a diverse set of roles in either restricting or modulating the lifecycles of select viruses. Here, we report that m6A levels are significantly increased in cells infected with the oncogenic human DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV). Transcriptome-wide m6A-sequencing of the KSHV-positive renal carcinoma cell line iSLK.219 during lytic reactivation revealed the presence of m6A across multiple kinetic classes of viral transcripts, and a concomitant decrease in m6A levels across much of the host transcriptome. However, we found that depletion of the m6A machinery had differential pro- and anti-viral impacts on viral gene expression depending on the cell-type analyzed. In iSLK.219 and iSLK.BAC16 cells the pathway functioned in a pro-viral manner, as depletion of the m6A writer METTL3 and the reader YTHDF2 significantly impaired virion production. In iSLK.219 cells the defect was linked to their roles in the post-transcriptional accumulation of the major viral lytic transactivator ORF50, which is m6A modified. In contrast, although the ORF50 mRNA was also m6A modified in KSHV infected B cells, ORF50 protein expression was instead increased upon depletion of METTL3, or, to a lesser extent, YTHDF2. These results highlight that the m6A pathway is centrally involved in regulating KSHV gene expression, and underscore how the outcome of this dynamically regulated modification can vary significantly between cell types.

Adenosine-to-inosine RNA editing meets cancer

Abstract: 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.

N6-methyladenosine-dependent regulation of messenger RNA stability

Abstract: N(6)-methyladenosine (m(6)A) is the most prevalent internal (non-cap) modification present in the messenger RNA of all higher eukaryotes. Although essential to cell viability and development, the exact role of m(6)A modification remains to be determined. The recent discovery of two m(6)A demethylases in mammalian cells highlighted the importance of m(6)A in basic biological functions and disease. Here we show that m(6)A is selectively recognized by the human YTH domain family 2 (YTHDF2) 'reader' protein to regulate mRNA degradation. We identified over 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs, but which also include non-coding RNAs, with a conserved core motif of G(m(6)A)C. We further establish the role of YTHDF2 in RNA metabolism, showing that binding of YTHDF2 results in the localization of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies. The carboxy-terminal domain of YTHDF2 selectively binds to m(6)A-containing mRNA, whereas the amino-terminal domain is responsible for the localization of the YTHDF2-mRNA complex to cellular RNA decay sites. Our results indicate that the dynamic m(6)A modification is recognized by selectively binding proteins to affect the translation status and lifetime of mRNA.

A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation

Abstract: Certain adenosine residues within mammalian RNAs undergo reversible N6 methylation. Two methyltransferase enzymes, METTL3 and METTL14, as well as the splicing factor WTAP are identified as core components of the multiprotein complex that deposits RNA N6-methyladenosine (m6A) in nuclear RNAs.