Unique Features and Anti-microbial Targeting of Folate- and Flavin-Dependent Methyltransferases Required for Accurate Maintenance of Genetic Information.

Comparative genome analyses have led to the discovery and characterization of novel flavin- and folate-dependent methyltransferases that mainly function in DNA precursor synthesis and post-transcriptional RNA modification by forming (ribo) thymidylate and its derivatives. Here we discuss the recent literature on the novel mechanistic features of these enzymes sometimes referred to as "uracil methyltransferases," albeit we prefer to refer to them as (ribo) thymidylate synthases. These enzyme families attest to the convergent evolution of nucleic acid methylation. Special focus is given to describing the unique characteristics of these flavin- and folate-dependent enzymes that have emerged as new models for studying the non-canonical roles of reduced flavin co-factors (FADH2) in relaying carbon atoms between enzyme substrates. This ancient enzymatic methylation mechanism with a very wide phylogenetic distribution may be more commonly used for biological methylation reactions than previously anticipated. This notion is exemplified by the recent discovery of additional substrates for these enzymes. Moreover, similar reaction mechanisms can be reversed by demethylases, which remove methyl groups e.g., from human histones. Future work is now required to address whether the use of different methyl donors facilitates the regulation of distinct methylation reactions in the cell. It will also be of great interest to address whether the low activity flavin-dependent thymidylate synthases ThyX represent ancestral enzymes that were eventually replaced by the more active thymidylate synthases of the ThyA family to facilitate the maintenance of larger genomes in fast-growing microbes. Moreover, we discuss the recent efforts from several laboratories to identify selective anti-microbial compounds that target flavin-dependent thymidylate synthase ThyX. Altogether we underline how the discovery of the alternative flavoproteins required for methylation of DNA and/or RNA nucleotides, in addition to providing novel targets for antibiotics, has provided new insight into microbial physiology and virulence.

emMAW: computing minimal absent words in external memory.

MOTIVATION: The biological significance of minimal absent words has been investigated in genomes of organisms from all domains of life. For instance, three minimal absent words of the human genome were found in Ebola virus genomes. There exists an O(n) -time and O(n) -space algorithm for computing all minimal absent words of a sequence of length n on a fixed-sized alphabet based on suffix arrays. A standard implementation of this algorithm, when applied to a large sequence of length n , requires more than 20 n  bytes of RAM. Such memory requirements are a significant hurdle to the computation of minimal absent words in large datasets. RESULTS: We present emMAW, the first external-memory algorithm for computing minimal absent words. A free open-source implementation of our algorithm is made available. This allows for computation of minimal absent words on far bigger data sets than was previously possible. Our implementation requires less than 3 h on a standard workstation to process the full human genome when as little as 1 GB of RAM is made available. We stress that our implementation, despite making use of external memory, is fast; indeed, even on relatively smaller datasets when enough RAM is available to hold all necessary data structures, it is less than two times slower than state-of-the-art internal-memory implementations. AVAILABILITY AND IMPLEMENTATION: https://github.com/solonas13/maw (free software under the terms of the GNU GPL). CONTACT: alice.heliou@lix.polytechnique.fr or solon.pissis@kcl.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

High-throughput sequencing reveals circular substrates for an archaeal RNA ligase.

It is only recently that the abundant presence of circular RNAs (circRNAs) in all kingdoms of Life, including the hyperthermophilic archaeon Pyrococcus abyssi, has emerged. This led us to investigate the physiologic significance of a previously observed weak intramolecular ligation activity of Pab1020 RNA ligase. Here we demonstrate that this enzyme, despite sharing significant sequence similarity with DNA ligases, is indeed an RNA-specific polynucleotide ligase efficiently acting on physiologically significant substrates. Using a combination of RNA immunoprecipitation assays and RNA-seq, our genome-wide studies revealed 133 individual circRNA loci in P. abyssi. The large majority of these loci interacted with Pab1020 in cells and circularization of selected C/D Box and 5S rRNA transcripts was confirmed biochemically. Altogether these studies revealed that Pab1020 is required for RNA circularization. Our results further suggest the functional speciation of an ancestral NTase domain and/or DNA ligase toward RNA ligase activity and prompt for further characterization of the widespread functions of circular RNAs in prokaryotes. Detailed insight into the cellular substrates of Pab1020 may facilitate the development of new biotechnological applications e.g. in ligation of preadenylated adaptors to RNA molecules.

Linear-time computation of minimal absent words using suffix array.

BACKGROUND: An absent word of a word y of length n is a word that does not occur in y. It is a minimal absent word if all its proper factors occur in y. Minimal absent words have been computed in genomes of organisms from all domains of life; their computation also provides a fast alternative for measuring approximation in sequence comparison. There exists an [Formula: see text]-time and [Formula: see text]-space algorithm for computing all minimal absent words on a fixed-sized alphabet based on the construction of suffix automata (Crochemore et al., 1998). No implementation of this algorithm is publicly available. There also exists an [Formula: see text]-time and [Formula: see text]-space algorithm for the same problem based on the construction of suffix arrays (Pinho et al., 2009). An implementation of this algorithm was also provided by the authors and is currently the fastest available. RESULTS: Our contribution in this article is twofold: first, we bridge this unpleasant gap by presenting an [Formula: see text]-time and [Formula: see text]-space algorithm for computing all minimal absent words based on the construction of suffix arrays; and second, we provide the respective implementation of this algorithm. Experimental results, using real and synthetic data, show that this implementation outperforms the one by Pinho et al. The open-source code of our implementation is freely available at http://github.com/solonas13/maw . CONCLUSIONS: Classical notions for sequence comparison are increasingly being replaced by other similarity measures that refer to the composition of sequences in terms of their constituent patterns. One such measure is the minimal absent words. In this article, we present a new linear-time and linear-space algorithm for the computation of minimal absent words based on the suffix array.