Identification of direct targets and modified bases of RNA cytosine methyltransferases
TL;DR: When applied in a human cell line to the RNA methyltransferases DNMT2 and NSUN2, Aza-IP enabled >200-fold enrichment of tRNAs that are known targets of the enzymes and revealed many tRNA and noncoding RNA targets not previously associated withNSUN2.
Abstract: Covalent links formed between methylation enzymes and a 5-azacytidine base incorporated into cellular RNA allow target enrichment and single base-pair resolution modification mapping.
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01 Jan 2011
TL;DR: The sheer volume and scope of data posed by this flood of data pose a significant challenge to the development of efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data.
Abstract: Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.
2,187 citations
TL;DR: Roles for mRNA modification in nearly every aspect of the mRNA life cycle, as well as in various cellular, developmental, and disease processes are revealed.
1,855 citations
Cites background or methods from "Identification of direct targets an..."
...…NSUN2 has been identified as the methyltransferase responsible for m5C methylation in several mRNAs and lncRNAs (Hussain et al., 2013; Khoddami and Cairns, 2013)). m5C is recognized by the mRNA export adaptor protein ALYREF, suggesting a role in nuclear export of m5C-containing…...
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...The tRNA m(5)C methyltransferase NSUN2 has been identified as the methyltransferase responsible for m(5)C methylation in several mRNAs and lncRNAs (Hussain et al., 2013; Khoddami and Cairns, 2013))....
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...…of m5C in nearly 300 mRNAs by miCLIP (Hussain et al., 2013), though fewer coding transcripts were identified as targets using other methods (Khoddami and Cairns, 2013; Squires et al., 2012). m5C can be oxidized in Drosophila by a conserved Tet ortholog CG43444 (dTet) to generate hm5C in…...
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..., 2013), though fewer coding transcripts were identified as targets using other methods (Khoddami and Cairns, 2013; Squires et al., 2012)....
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TL;DR: N6-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses.
Abstract: The recent discovery of reversible mRNA methylation has opened a new realm of post-transcriptional gene regulation in eukaryotes. The identification and functional characterization of proteins that specifically recognize RNA N6-methyladenosine (m6A) unveiled it as a modification that cells utilize to accelerate mRNA metabolism and translation. N6-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses. Other mRNA modifications, including N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine, together with m6A form the epitranscriptome and collectively code a new layer of information that controls protein synthesis.
1,369 citations
TL;DR: It is shown 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.
Abstract: 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.
678 citations
TL;DR: It is revealed that m5C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes.
Abstract: 5-methylcytosine (m5C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m5C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m5C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m5C profiles of mammalian transcriptomes and suggests an essential role for m5C modification in mRNA export and post-transcriptional regulation.
543 citations
References
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TL;DR: SAMtools as discussed by the authors implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments.
Abstract: Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments.
Availability: http://samtools.sourceforge.net
Contact: [email protected]
45,957 citations
TL;DR: The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large by making use of universally available web GUIs (Graphical User Interfaces).
Abstract: The abbreviated name,‘mfold web server’,describes a number of closely related software applications available on the World Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making use of universally available web GUIs (Graphical User Interfaces),the server circumvents the problem of portability of this software. Detailed output,in the form of structure plots with or without reliability information,single strand frequency plots and ‘energy dot plots’, are available for the folding of single sequences. A variety of ‘bulk’ servers give less information,but in a shorter time and for up to hundreds of sequences at once. The portal for the mfold web server is http://www.bioinfo.rpi.edu/applications/ mfold. This URL will be referred to as ‘MFOLDROOT’.
12,535 citations
TL;DR: In this article, the authors present an approach for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data.
Abstract: Rapid improvements in sequencing and array-based platforms are resulting in a flood of diverse genome-wide data, including data from exome and whole-genome sequencing, epigenetic surveys, expression profiling of coding and noncoding RNAs, single nucleotide polymorphism (SNP) and copy number profiling, and functional assays. Analysis of these large, diverse data sets holds the promise of a more comprehensive understanding of the genome and its relation to human disease. Experienced and knowledgeable human review is an essential component of this process, complementing computational approaches. This calls for efficient and intuitive visualization tools able to scale to very large data sets and to flexibly integrate multiple data types, including clinical data. However, the sheer volume and scope of data pose a significant challenge to the development of such tools.
10,798 citations
TL;DR: Disaggregated mouse embryo cells, grown in monolayers, underwent a progressive decline in growth rate upon successive transfer, the rapidity of the decline depending on the inoculation density, but nearly all cultures developed into established lines within 3 months of culture.
Abstract: Disaggregated mouse embryo cells, grown in monolayers, underwent a progressive decline in growth rate upon successive transfer, the rapidity of the decline depending, among other things, on the inoculation density. Nevertheless, nearly all cultures developed into established lines within 3 months of culture. The first sign of the emergence of an established line was the ability of the cells to maintain a constant or rising potential growth rate. This occurred while the cultures were morphologically unchanged. The growth rate continued to increase until it equaled or exceeded that of the original culture. The early established cells showed an increasing metabolic autonomy, as indicated by decreasing dependence on cell-to-cell feeding. It is suggested that the process of establishment involves an alteration in cell permeability properties. Chromosome studies indicated that the cells responsible for the upturn in growth rate were diploid, but later the population shifted to the tetraploid range, often very rapidly. Still later, marker chromosomes appeared. Different lines acquired different properties, depending on the culture conditions employed; one line developed which is extremely sensitive to contact inhibition.
2,654 citations
TL;DR: A computer program, ARAGORN, identifies tRNA and tmRNA genes using heuristic algorithms to predict tRNA secondary structure, based on homology with recognized tRNA consensus sequences and ability to form a base-paired cloverleaf.
Abstract: A computer program, ARAGORN, identifies tRNA and tmRNA genes. The program employs heuristic algorithms to predict tRNA secondary structure, based on homology with recognized tRNA consensus sequences and ability to form a base-paired cloverleaf. tmRNA genes are identified using a modified version of the BRUCE program. ARAGORN achieves a detection sensitivity of 99% from a set of 1290 eubacterial, eukaryotic and archaeal tRNA genes and detects all complete tmRNA sequences in the tmRNA database, improving on the performance of the BRUCE program. Recently discovered tmRNA genes in the chloroplasts of two species from the 'green' algae lineage are detected. The output of the program reports the proposed tRNA secondary structure and, for tmRNA genes, the secondary structure of the tRNA domain, the tmRNA gene sequence, the tag peptide and a list of organisms with matching tmRNA peptide tags.
1,964 citations