The 5-methylcytosine (5-mC) derivative 5-hydroxymethylcytosine (5-hmC) is abundant in the brain for unknown reasons. Here we characterize the genomic distribution of 5-hmC and 5-mC in human and mouse ...tissues. We assayed 5-hmC by using glucosylation coupled with restriction-enzyme digestion and microarray analysis. We detected 5-hmC enrichment in genes with synapse-related functions in both human and mouse brain. We also identified substantial tissue-specific differential distributions of these DNA modifications at the exon-intron boundary in human and mouse. This boundary change was mainly due to 5-hmC in the brain but due to 5-mC in non-neural contexts. This pattern was replicated in multiple independent data sets and with single-molecule sequencing. Moreover, in human frontal cortex, constitutive exons contained higher levels of 5-hmC relative to alternatively spliced exons. Our study suggests a new role for 5-hmC in RNA splicing and synaptic function in the brain.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Guanosines are important for biological activities through their specific functional groups that are recognized for RNA or protein interactions. One example is recognition of N(1) of G37 in tRNA by ...S-adenosyl-methionine (AdoMet)-dependent tRNA methyltransferases to synthesize m(1)G37-tRNA, which is essential for translational fidelity in all biological domains. Synthesis of m(1)G37-tRNA is catalyzed by TrmD in bacteria and by Trm5 in eukarya and archaea, using unrelated and dissimilar structural folds. This raises the question of how dissimilar proteins recognize the same guanosine. Here we probe the mechanism of discrimination among functional groups of guanosine by TrmD and Trm5. Guanosine analogs were systematically introduced into tRNA through a combination of chemical and enzymatic synthesis. Single turnover kinetic assays and thermodynamic analysis of the effect of each analog on m(1)G37-tRNA synthesis reveal that TrmD and Trm5 discriminate functional groups differently. While both recognize N(1) and O(6) of G37, TrmD places a much stronger emphasis on these functional groups than Trm5. While the exocyclic 2-amino group of G37 is important for TrmD, it is dispensable for Trm5. In addition, while an adjacent G36 is obligatory for TrmD, it is nonessential for Trm5. These results depict a more rigid requirement of guanosine functional groups for TrmD than for Trm5. However, the sensitivity of both enzymes to analog substitutions, together with an experimental revelation of their low cellular concentrations relative to tRNA substrates, suggests a model in which these enzymes rapidly screen tRNA by direct recognition of G37 in order to monitor the global state of m(1)G37-tRNA.
Epigenetic phenomena play a central role in cell regulatory processes and are important factors for understanding complex human disease. One of the best understood epigenetic mechanisms is DNA ...methylation. In the mammalian genome, cytosines (C) in CpG dinucleotides were long known to undergo methylation at the 5-position of the pyrimidine ring (mC). Later it was found that mC can be oxidized to 5-hydroxymethylcytosine (hmC) or even further to 5-formylcytosine (fC) and to 5-carboxylcytosine (caC) by the action of 2-oxoglutarate-dependent dioxygenases of the TET family. These findings unveiled a long elusive mechanism of active DNA demethylation and bolstered a wave of studies in the area of epigenetic regulation in mammals. This review is dedicated to critical assessment of recent data on biochemical and chemical aspects of the formation and conversion of hmC in DNA, analytical techniques used for detection and mapping of this nucleobase in mammalian genomes as well as epigenetic roles of hmC in DNA replication, transcription, cell differentiation and human disease.
This review assesses current analytical approaches and the mechanistic, biological and biomedical significance of 5-hydroxymethylcytosine - the second most abundant modified base in mammalian DNA.
ConspectusDNA is the genetic matter of life composed of four major nucleotides which can be further furnished with biologically important covalent modifications. Among the variety of enzymes involved ...in DNA metabolism, AdoMet-dependent methyltransferases (MTases) combine the recognition of specific sequences and covalent methylation of a target nucleotide. The naturally transferred methyl groups play important roles in biological signaling, but they are poor physical reporters and largely resistant to chemical derivatization. Therefore, an obvious strategy to unlock the practical utility of the methyltransferase reactions is to enable the transfer of "prederivatized" (extended) versions of the methyl group.However, previous enzymatic studies of extended AdoMet analogs indicated that the transalkylation reactions are drastically impaired as the size of the carbon chain increases. In collaborative efforts, we proposed that, akin to enhanced S
2 reactivity of allylic and propargylic systems, addition of a π orbital next to the transferable carbon atom might confer the needed activation of the reaction. Indeed, we found that MTase-catalyzed transalkylations of DNA with cofactors containing a double or a triple C-C bond in the β position occurred in a robust and sequence-specific manner. Altogether, this breakthrough approach named mTAG (methyltransferase-directed transfer of activated groups) has proven instrumental for targeted labeling of DNA and other types of biomolecules (using appropriate MTases) including RNA and proteins.Our further work focused on the propargylic cofactors and their reactions with DNA cytosine-5 MTases, a class of MTases common for both prokaryotes and eukaryotes. Here, we learned that the 4-X-but-2-yn-1-yl (X = polar group) cofactors suffered from a rapid loss of activity in aqueous buffers due to susceptibility of the triple bond to hydration. This problem was remedied by synthetically increasing the separation between X and the triple bond from one to three carbon units (6-X-hex-2-ynyl cofactors). To further optimize the transfer of the bulkier groups, we performed structure-guided engineering of the MTase cofactor pocket. Alanine replacements of two conserved residues conferred substantial improvements of the transalkylation activity with M.HhaI and three other engineered bacterial C5-MTases. Of particular interest were CpG-specific DNA MTases (M.SssI), which proved valuable tools for studies of mammalian methylomes and chemical probing of DNA function.Inspired by the successful repurposing of bacterial enzymes, we turned to more complex mammalian C5-MTases (Dnmt1, Dnmt3A, and Dnmt3B) and asked if they could ultimately lead to mTAG labeling inside mammalian cells. Our efforts to engineer mouse Dnmt1 produced a variant (Dnmt1*) that enabled efficient Dnmt1-directed deposition of 6-azide-hexynyl groups on DNA in vitro. CRISPR-Cas9 editing of the corresponding codons in the genomic Dnmt1 alleles established endogenous expression of Dnmt1* in mouse embryonic stem cells. To circumvent the poor cellular uptake of AdoMet and its analogs, we elaborated their efficient internalization by electroporation, which has finally enabled selective catalysis-dependent azide tagging of natural Dnmt1 targets in live mammalian cells. The deposited chemical groups were then exploited as "click" handles for reading adjoining sequences and precise genomic mapping of the methylation sites. These findings offer unprecedented inroads into studies of DNA methylation in a wide range of eukaryotic model systems.
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•Enzymatic transfer of chemically extended groups enables targeted covalent functionalization of DNA and RNA.•Methyltransferase-directed labeling of DNA and RNA provides novel tools ...for (epi)genomic and (epi)transcriptomic studies.•Detection of individual covalently-bound reporters on DNA permits single-molecule analysis of (epi)genomes.
Produced as linear biopolymers from four major types of building blocks, DNA and RNA are further furnished with a range of covalent modifications. Despite the impressive specificity of natural enzymes, the transferred groups are often poor reporters and not amenable to further derivatization. Therefore, strategies based on repurposing some of these enzymatic reactions to accept derivatized versions of the transferrable groups have been exploited. By far the most widely used are S-adenosylmethionine-dependent methyltransferases, which along with several other nucleic acids modifying enzymes offer a broad selection of tagging chemistries and molecular features on DNA and RNA that can be targeted in vitro and in vivo. Engineered enzymatic reactions have been implemented in validated DNA sequencing-based protocols for epigenome analysis. The utility of chemo-enzymatic labeling is further enhanced with recent advances in physical detection of individual reporter groups on DNA using super resolution microscopy and nanopore sensing enabling single-molecule multiplex analysis of genetic and epigenetic marks in minute samples. Altogether, a number of new powerful techniques are currently in use or on the verge of real benchtop applications as research tools or next generation diagnostics.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
RNA cleavage by bacterial RNA polymerase (RNAP) has been implicated in transcriptional proofreading and reactivation of arrested transcription elongation complexes but its molecular mechanism is less ...understood than the mechanism of nucleotide addition, despite both reactions taking place in the same active site. RNAP from the radioresistant bacterium Deinococcus radiodurans is characterized by highly efficient intrinsic RNA cleavage in comparison with Escherichia coli RNAP. We find that the enhanced RNA cleavage activity largely derives from amino acid substitutions in the trigger loop (TL), a mobile element of the active site involved in various RNAP activities. The differences in RNA cleavage between these RNAPs disappear when the TL is deleted, or in the presence of GreA cleavage factors, which replace the TL in the active site. We propose that the TL substitutions modulate the RNA cleavage activity by altering the TL folding and its contacts with substrate RNA and that the resulting differences in transcriptional proofreading may play a role in bacterial stress adaptation.
MicroRNAs regulate gene expression in numerous biological pathways and are typically methylated at their 3′-termini in plants but not in animals. Here we show that the HEN1 RNA 2′-O-methyltransferase ...from Arabidopsis thaliana catalyzes the transfer of extended propargylic moieties from synthetic AdoMet cofactor analogs to duplex miRNAs or siRNAs. The presented approach permits selective and efficient covalent labeling of small RNA duplexes with a variety of functional or reporter groups for their enrichment and analysis.
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S-Adenosylmethionine-dependent DNA methyltransferases (MTases) perform direct methylation of cytosine to yield 5-methylcytosine (5mC), which serves as part of the epigenetic regulation mechanism in ...vertebrates. Active demethylation of 5mC by TET oxygenases produces 5-formylcytosine (fC) and 5-carboxylcytosine (caC), which were shown to be enzymatically excised and then replaced with an unmodified nucleotide. Here we find that both bacterial and mammalian C5-MTases can catalyze the direct decarboxylation of caC yielding unmodified cytosine in DNA in vitro but are inert toward fC. The observed atypical enzymatic C–C bond cleavage reaction provides a plausible precedent for a direct reversal of caC to the unmodified state in DNA and offers a unique approach for sequence-specific analysis of genomic caC.
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Deep-sea inspirations to burning scientific passions, along with training by a Nobel Prize recipient, we talk to first author Vaidotas Stankevičius and group leaders, Giedrius Vilkaitis and Saulius ...Klimašauskas, about their paper, “Selective chemical tracking of Dnmt1 catalytic activity in live cells,” and the developing research landscape in Lithuania.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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