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1.  MODOMICS: a database of RNA modification pathways—2013 update 
Nucleic Acids Research  2012;41(D1):D262-D267.
MODOMICS is a database of RNA modifications that provides comprehensive information concerning the chemical structures of modified ribonucleosides, their biosynthetic pathways, RNA-modifying enzymes and location of modified residues in RNA sequences. In the current database version, accessible at http://modomics.genesilico.pl, we included new features: a census of human and yeast snoRNAs involved in RNA-guided RNA modification, a new section covering the 5′-end capping process, and a catalogue of ‘building blocks’ for chemical synthesis of a large variety of modified nucleosides. The MODOMICS collections of RNA modifications, RNA-modifying enzymes and modified RNAs have been also updated. A number of newly identified modified ribonucleosides and more than one hundred functionally and structurally characterized proteins from various organisms have been added. In the RNA sequences section, snRNAs and snoRNAs with experimentally mapped modified nucleosides have been added and the current collection of rRNA and tRNA sequences has been substantially enlarged. To facilitate literature searches, each record in MODOMICS has been cross-referenced to other databases and to selected key publications. New options for database searching and querying have been implemented, including a BLAST search of protein sequences and a PARALIGN search of the collected nucleic acid sequences.
doi:10.1093/nar/gks1007
PMCID: PMC3531130  PMID: 23118484
2.  Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria 
Nucleic Acids Research  2009;38(5):1652-1663.
The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase RlmN that methylates the C-2 position. Database searches with the Cfr sequence have revealed a large group of closely related sequences from all domains of life that contain the conserved CX3CX2C motif characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. Phylogenetic analysis of the Cfr/RlmN family suggests that the RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer. A structural model of Cfr has been calculated and used as a guide for alanine mutagenesis studies that corroborate the model-based predictions of a 4Fe–4S cluster, a SAM molecule coordinated to the iron–sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis. The investigation has identified essential amino acids and Cfr variants with altered reaction mechanisms and represents a first step towards understanding the structural basis of Cfr activity.
doi:10.1093/nar/gkp1142
PMCID: PMC2836569  PMID: 20007606
3.  MODOMICS: a database of RNA modification pathways. 2008 update 
Nucleic Acids Research  2008;37(Database issue):D118-D121.
MODOMICS, a database devoted to the systems biology of RNA modification, has been subjected to substantial improvements. It provides comprehensive information on the chemical structure of modified nucleosides, pathways of their biosynthesis, sequences of RNAs containing these modifications and RNA-modifying enzymes. MODOMICS also provides cross-references to other databases and to literature. In addition to the previously available manually curated tRNA sequences from a few model organisms, we have now included additional tRNAs and rRNAs, and all RNAs with 3D structures in the Nucleic Acid Database, in which modified nucleosides are present. In total, 3460 modified bases in RNA sequences of different organisms have been annotated. New RNA-modifying enzymes have been also added. The current collection of enzymes includes mainly proteins for the model organisms Escherichia coli and Saccharomyces cerevisiae, and is currently being expanded to include proteins from other organisms, in particular Archaea and Homo sapiens. For enzymes with known structures, links are provided to the corresponding Protein Data Bank entries, while for many others homology models have been created. Many new options for database searching and querying have been included. MODOMICS can be accessed at http://genesilico.pl/modomics.
doi:10.1093/nar/gkn710
PMCID: PMC2686465  PMID: 18854352
4.  Functional specialization of domains tandemly duplicated within 16S rRNA methyltransferase RsmC 
Nucleic Acids Research  2007;35(13):4264-4274.
RNA methyltransferases (MTases) are important players in the biogenesis and regulation of the ribosome, the cellular machine for protein synthesis. RsmC is a MTase that catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (SAM) to G1207 of 16S rRNA. Mutations of G1207 have dominant lethal phenotypes in Escherichia coli, underscoring the significance of this modified nucleotide for ribosome function. Here we report the crystal structure of E. coli RsmC refined to 2.1 Å resolution, which reveals two homologous domains tandemly duplicated within a single polypeptide. We characterized the function of the individual domains and identified key residues involved in binding of rRNA and SAM, and in catalysis. We also discovered that one of the domains is important for the folding of the other. Domain duplication and subfunctionalization by complementary degeneration of redundant functions (in particular substrate binding versus catalysis) has been reported for many enzymes, including those involved in RNA metabolism. Thus, RsmC can be regarded as a model system for functional streamlining of domains accompanied by the development of dependencies concerning folding and stability.
doi:10.1093/nar/gkm411
PMCID: PMC1934991  PMID: 17576679
5.  Structural and evolutionary bioinformatics of the SPOUT superfamily of methyltransferases 
BMC Bioinformatics  2007;8:73.
Background
SPOUT methyltransferases (MTases) are a large class of S-adenosyl-L-methionine-dependent enzymes that exhibit an unusual alpha/beta fold with a very deep topological knot. In 2001, when no crystal structures were available for any of these proteins, Anantharaman, Koonin, and Aravind identified homology between SpoU and TrmD MTases and defined the SPOUT superfamily. Since then, multiple crystal structures of knotted MTases have been solved and numerous new homologous sequences appeared in the databases. However, no comprehensive comparative analysis of these proteins has been carried out to classify them based on structural and evolutionary criteria and to guide functional predictions.
Results
We carried out extensive searches of databases of protein structures and sequences to collect all members of previously identified SPOUT MTases, and to identify previously unknown homologs. Based on sequence clustering, characterization of domain architecture, structure predictions and sequence/structure comparisons, we re-defined families within the SPOUT superfamily and predicted putative active sites and biochemical functions for the so far uncharacterized members. We have also delineated the common core of SPOUT MTases and inferred a multiple sequence alignment for the conserved knot region, from which we calculated the phylogenetic tree of the superfamily. We have also studied phylogenetic distribution of different families, and used this information to infer the evolutionary history of the SPOUT superfamily.
Conclusion
We present the first phylogenetic tree of the SPOUT superfamily since it was defined, together with a new scheme for its classification, and discussion about conservation of sequence and structure in different families, and their functional implications. We identified four protein families as new members of the SPOUT superfamily. Three of these families are functionally uncharacterized (COG1772, COG1901, and COG4080), and one (COG1756 represented by Nep1p) has been already implicated in RNA metabolism, but its biochemical function has been unknown. Based on the inference of orthologous and paralogous relationships between all SPOUT families we propose that the Last Universal Common Ancestor (LUCA) of all extant organisms contained at least three SPOUT members, ancestors of contemporary RNA MTases that carry out m1G, m3U, and 2'O-ribose methylation, respectively. In this work we also speculate on the origin of the knot and propose possible 'unknotted' ancestors. The results of our analysis provide a comprehensive 'roadmap' for experimental characterization of SPOUT MTases and interpretation of functional studies in the light of sequence-structure relationships.
doi:10.1186/1471-2105-8-73
PMCID: PMC1829167  PMID: 17338813
6.  The yfhQ gene of Escherichia coli encodes a tRNA:Cm32/Um32 methyltransferase 
Background
Naturally occurring tRNAs contain numerous modified nucleosides. They are formed by enzymatic modification of the primary transcripts during the complex RNA maturation process. In model organisms Escherichia coli and Saccharomyces cerevisiae most enzymes involved in this process have been identified. Interestingly, it was found that tRNA methylation, one of the most common modifications, can be introduced by S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases (MTases) that belong to two structurally and phylogenetically unrelated protein superfamilies: RFM and SPOUT.
Results
As a part of a large-scale project aiming at characterization of a complete set of RNA modification enzymes of model organisms, we have studied the Escherichia coli proteins YibK, LasT, YfhQ, and YbeA for their ability to introduce the last unassigned methylations of ribose at positions 32 and 34 of the tRNA anticodon loop. We found that YfhQ catalyzes the AdoMet-dependent formation of Cm32 or Um32 in tRNASer1 and tRNAGln2 and that an E. coli strain with a disrupted yfhQ gene lacks the tRNA:Cm32/Um32 methyltransferase activity. Thus, we propose to rename YfhQ as TrMet(Xm32) according to the recently proposed, uniform nomenclature for all RNA modification enzymes, or TrmJ, according to the traditional nomenclature for bacterial tRNA MTases.
Conclusion
Our results reveal that methylation at position 32 is carried out by completely unrelated TrMet(Xm32) enzymes in eukaryota and prokaryota (RFM superfamily member Trm7 and SPOUT superfamily member TrmJ, respectively), mirroring the scenario observed in the case of the m1G37 modification (introduced by the RFM member Trm5 in eukaryota and archaea, and by the SPOUT member TrmD in bacteria).
doi:10.1186/1471-2199-7-23
PMCID: PMC1569432  PMID: 16848900
7.  Structural analysis of human 2′-O-ribose methyltransferases involved in mRNA cap structure formation 
Nature Communications  2014;5:3004.
The 5′ cap of human messenger RNA contains 2′-O-methylation of the first and often second transcribed nucleotide that is important for its processing, translation and stability. Human enzymes that methylate these nucleotides, termed CMTr1 and CMTr2, respectively, have recently been identified. However, the structures of these enzymes and their mechanisms of action remain unknown. In the present study, we solve the crystal structures of the active CMTr1 catalytic domain in complex with a methyl group donor and a capped oligoribonucleotide, thereby revealing the mechanism of specific recognition of capped RNA. This mechanism differs significantly from viral enzymes, thus providing a framework for their specific targeting. Based on the crystal structure of CMTr1, a comparative model of the CMTr2 catalytic domain is generated. This model, together with mutational analysis, leads to the identification of residues involved in RNA and methyl group donor binding.
Human mRNA transcripts possess a 5' cap structure that is modified by methylation. Here, Smietanski et al. present the structures of human methyltransferases responsible for this reaction, revealing key differences to their viral counterparts and thereby providing a framework for targeted drug design.
doi:10.1038/ncomms4004
PMCID: PMC3941023  PMID: 24402442

Results 1-7 (7)