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Overcoming conservation in TALE-DNA interactions: a minimal repeat scaffold enables selective recognition of an oxidized 5-methylcytosine.

ABSTRACT: Transcription-activator-like effectors (TALEs) are repeat-based proteins featuring programmable DNA binding. The repulsion of TALE repeats by 5-methylcytosine (5mC) and its oxidized forms makes TALEs potential probes for their programmable analysis. However, this potential has been limited by the inability to engineer repeats capable of actual, fully selective binding of an (oxidized) 5mC: the extremely conserved and simple nucleobase recognition mode of TALE repeats and their extensive involvement in inter-repeat interactions that stabilize the TALE fold represent major engineering hurdles. We evaluated libraries of alternative, strongly truncated repeat scaffolds and discovered a repeat that selectively recognizes 5-carboxylcytosine (5caC), enabling construction of the first programmable receptors for an oxidized 5mC. In computational studies, this unusual scaffold executes a dual function via a critical arginine that provides inter-repeat stabilization and selectively interacts with the 5caC carboxyl group via a salt-bridge. These findings argue for an unexpected adaptability of TALE repeats and provide a new impulse for the design of programmable probes for nucleobases beyond A, G, T and C.

SUBMITTER: Maurer S

PROVIDER: S-EPMC6148557 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Overcoming conservation in TALE-DNA interactions: a minimal repeat scaffold enables selective recognition of an oxidized 5-methylcytosine.

Maurer Sara S Buchmuller Benjamin B Ehrt Christiane C Jasper Julia J Koch Oliver O Summerer Daniel D

Chemical science 20180802 36

Transcription-activator-like effectors (TALEs) are repeat-based proteins featuring programmable DNA binding. The repulsion of TALE repeats by 5-methylcytosine (5mC) and its oxidized forms makes TALEs potential probes for their programmable analysis. However, this potential has been limited by the inability to engineer repeats capable of actual, fully selective binding of an (oxidized) 5mC: the extremely conserved and simple nucleobase recognition mode of TALE repeats and their extensive involvem ...[more]

PMID: 30288245

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Project description:BackgroundPost-transcriptional methylation of RNA cytosine residues to 5-methylcytosine (m(5)C) is an important modification that regulates RNA metabolism and occurs in both eukaryotes and prokaryotes. Yet, to date, no transcriptome-wide identification of m(5)C sites has been undertaken in plants. Plants provide a unique comparative system for investigating the origin and evolution of m(5)C as they contain three different genomes, the nucleus, mitochondria and chloroplast. Here we use bisulfite conversion of RNA combined with high-throughput IIlumina sequencing (RBS-seq) to identify single-nucleotide resolution of m(5)C sites in non-coding ribosomal RNAs and transfer RNAs of all three sub-cellular transcriptomes across six diverse species that included, the single-celled algae Nannochloropsis oculata, the macro algae Caulerpa taxifolia and multi-cellular higher plants Arabidopsis thaliana, Brassica rapa, Triticum durum and Ginkgo biloba.ResultsUsing the plant model Arabidopsis thaliana, we identified a total of 39 highly methylated m(5)C sites in predicted structural positions of nuclear tRNAs and 7 m(5)C sites in rRNAs from nuclear, chloroplast and mitochondrial transcriptomes. Both the nucleotide position and percent methylation of tRNAs and rRNAs m(5)C sites were conserved across all species analysed, from single celled algae N. oculata to multicellular plants. Interestingly the mitochondrial and chloroplast encoded tRNAs were devoid of m(5)C in A. thaliana and this is generally conserved across Plantae. This suggests independent evolution of organelle methylation in animals and plants, as animal mitochondrial tRNAs have m(5)C sites. Here we characterize 5 members of the RNA 5-methylcytosine family in Arabidopsis and extend the functional characterization of TRDMT1 and NOP2A/OLI2. We demonstrate that nuclear tRNA methylation requires two evolutionarily conserved methyltransferases, TRDMT1 and TRM4B. trdmt1 trm4b double mutants are hypersensitive to the antibiotic hygromycin B, demonstrating the function of tRNA methylation in regulating translation. Additionally we demonstrate that nuclear large subunit 25S rRNA methylation requires the conserved RNA methyltransferase NSUN5. Our results also suggest functional redundancy of at least two of the NOP2 paralogs in Arabidopsis.ConclusionsOur data demonstrates widespread occurrence and conservation of non-coding RNA methylation in the kingdom Plantae, suggesting important and highly conserved roles of this post-transcriptional modification.

Project description:Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC) and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC) and adult mouse brain tissue. Readers for these modifications are only partially overlapping and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation. Peptides were separated on an EASY-nLC (Proxeon) connected online to an LTQ-Orbitrap-Velos mass spectrometer. Spectra were recorded in CID mode. A gradient of organic solvent (5-30% acetonitrile) was applied (120 minutes) and the top 15 most abundant peptides were fragmented for MS/MS, using an exclusion list of 500 proteins for 45 seconds. Raw data were analyzed using Maxquant version 1.2.2.5 and the integrated Andromeda search engine against protein database ipi.MOUSE.v3.68. Using Perseus, data was filtered for contaminants, reverse hits, number of peptides (>1) and unique peptides (>0). Ratios were logarithmitized (log2) and groups (consisting of forward and reverse) were defined. Proteins were filtered to have at least 2 valid values in one of the groups and missing values were imputed based on a normal distribution (width=0.2 and shift=0), after which Significance B was calculated (Benj.Hoch.FDR=0.05). Scatterplots were made using R. Proteins were defined to be significant when both forward and reverse significance p<0.05 and minimal ratios were >2 in both experiments. The H/L ratios shown in Figure2A-C were calculated using the formula (log(forward ratio) - log(reverse ratio))/2.

Dataset Information

Overcoming conservation in TALE-DNA interactions: a minimal repeat scaffold enables selective recognition of an oxidized 5-methylcytosine.

Publications

Overcoming conservation in TALE-DNA interactions: a minimal repeat scaffold enables selective recognition of an oxidized 5-methylcytosine.

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