R-Coffee: a method for multiple alignment of non-coding RNA

R-Coffee is a multiple RNA alignment package, derived from T-Coffee, designed to align RNA sequences while exploiting secondary structure information. R-Coffee uses an alignment-scoring scheme that incorporates secondary structure information within the alignment. It works particularly well as an alignment improver and can be combined with any existing sequence alignment method. In this work, we used R-Coffee to compute multiple sequence alignments combining the pairwise output of sequence aligners and structural aligners. We show that R-Coffee can improve the accuracy of all the sequence aligners. We also show that the consistency-based component of T-Coffee can improve the accuracy of several structural aligners. R-Coffee was tested on 388 BRAliBase reference datasets and on 11 longer Cmfinder datasets. Altogether our results suggest that the best protocol for aligning short sequences (less than 200 nt) is the combination of R-Coffee with the RNA pairwise structural aligner Consan. We also show that the simultaneous combination of the four best sequence alignment programs with R-Coffee produces alignments almost as accurate as those obtained with R-Coffee/Consan. Finally, we show that R-Coffee can also be used to align longer datasets beyond the usual scope of structural aligners. R-Coffee is freely available for download, along with documentation, from the T-Coffee web site (www.tcoffee.org)

Membrane Topology and Predicted RNA-Binding Function of the ‘Early Responsive to Dehydration (ERD4)’ Plant Protein

Functional annotation of uncharacterized genes is the main focus of computational methods in the post genomic era. These tools search for similarity between proteins on the premise that those sharing sequence or structural motifs usually perform related functions, and are thus particularly useful for membrane proteins. Early responsive to dehydration (ERD) genes are rapidly induced in response to dehydration stress in a variety of plant species. In the present work we characterized function of Brassica juncea ERD4 gene using computational approaches. The ERD4 protein of unknown function possesses ubiquitous DUF221 domain (residues 312–634) and is conserved in all plant species. We suggest that the protein is localized in chloroplast membrane with at least nine transmembrane helices. We detected a globular domain of 165 amino acid residues (183–347) in plant ERD4 proteins and expect this to be posited inside the chloroplast. The structural-functional annotation of the globular domain was arrived at using fold recognition methods, which suggested in its sequence presence of two tandem RNA-recognition motif (RRM) domains each folded into βαββαβ topology. The structure based sequence alignment with the known RNA-binding proteins revealed conservation of two non-canonical ribonucleoprotein sub-motifs in both the putative RNA-recognition domains of the ERD4 protein. The function of highly conserved ERD4 protein may thus be associated with its RNA-binding ability during the stress response. This is the first functional annotation of ERD4 family of proteins that can be useful in designing experiments to unravel crucial aspects of stress tolerance mechanism

TI2BioP — Topological Indices to BioPolymers. A Graphical– Numerical Approach for Bioinformatics

We developed a new graphical–numerical method called TI2BioP (Topological Indices to BioPolymers) to estimate topological indices (TIs) from two-dimensional (2D) graphical approaches for the natural biopolymers DNA, RNA and proteins The methodology mainly turns long biopolymeric sequences into 2D artificial graphs such as Cartesian and four-color maps but also reads other 2D graphs from the thermodynamic folding of DNA/RNA strings inferred from other programs. The topology of such 2D graphs is either encoded by node or adjacency matrixes for the calculation of the spectral moments as TIs. These numerical indices were used to build up alignment-free models to the functional classification of biosequences and to calculate alignment-free distances for phylogenetic purposes. The performance of the method was evaluated in highly diverse gene/protein classes, which represents a challenge for current bioinformatics algorithms. TI2BioP generally outperformed classical bioinformatics algorithms in the functional classification of Bacteriocins, ribonucleases III (RNases III), genomic internal transcribed spacer II (ITS2) and adenylation domains (A-domains) of nonribosomal peptide synthetases (NRPS) allowing the detection of new members in these target gene/protein classes. TI2BioP classification performance was contrasted and supported by predictions with sensitive alignment-based algorithms and experimental outcomes, respectively. The new ITS2 sequence isolated from Petrakia sp. was used in our graphical–numerical approach to estimate alignment-free distances for phylogenetic inferences. Despite TI2BioP having been developed for application in bioinformatics, it can be extended to predict interesting features of other biopolymers than DNA and protein sequences. TI2BioP version 2.0 is freely available from http://ti2biop.sourceforge.net/

Evolutionary and structural aspects of Solanaceae RNases T2

Plant RNases T2 are involved in several physiological and developmental processes, including inorganic phosphate starvation, senescence, wounding, defense against pathogens, and the self-incompatibility system. Solanaceae RNases form three main clades, one composed exclusively of S-RNases and two that include S-like RNases. We identified several positively selected amino acids located in highly flexible regions of these molecules, mainly close to the B1 and B2 substrate-binding sites in S-like RNases and the hypervariable regions of S-RNases. These differences between S- and S-like RNases in the flexibility of amino acids in substrate-binding regions are essential to understand the RNA-binding process. For example, in the S-like RNase NT, two positively selected amino acid residues (Tyr156 and Asn134) are located at the most flexible sites on the molecular surface. RNase NT is induced in response to tobacco mosaic virus infection; these sites may thus be regions of interaction with pathogen proteins or viral RNA. Differential selective pressures acting on plant ribonucleases have increased amino acid variability and, consequently, structural differences within and among S-like RNases and S-RNases that seem to be essential for these proteins play different functions

The MAP kinase HwHog1 from the halophilic black yeast Hortaea werneckii: coping with stresses in solar salterns

BACKGROUND: Hortaea werneckii is one of the most salt-tolerant species among microorganisms. It has been isolated from hypersaline waters of salterns as one of the predominant species of a group of halophilic and halotolerant melanized yeast-like fungi, arbitrarily named as "black yeasts". It has previously been shown that H. werneckii has distinct mechanisms of adaptation to high salinity environments that are not seen in salt-sensitive and only moderately salt-tolerant fungi. In H. werneckii, the HOG pathway is important for sensing the changes in environmental osmolarity, as demonstrated by identification of three main pathway components: the mitogen-activated protein kinase (MAPK) HwHog1, the MAPK kinase HwPbs2, and the putative histidine kinase osmosensor HwHhk7. RESULTS: In this study, we show that the expression of HwHOG1 in salt-adapted cells depends on the environmental salinity and that HwHOG1 transcription responds rapidly but reciprocally to the acute hyper-saline or hypo-saline stress. Molecular modelling of HwHog1 reveals an overall structural homology with other MAPKs. HwHog1 complements the function of ScHog1 in the Saccharomyces cerevisiae multistress response. We also show that hyper-osmolar, oxidative and high-temperature stresses activate the HwHog1 kinase, although under high-temperature stress the signal is not transmitted via the MAPK kinase Pbs2. Identification of HOG1-like genes from other halotolerant fungi isolated from solar salterns demonstrates a high degree of similarity and excellent phylogenetic clustering with orthologues of fungal origin. CONCLUSION: The HOG signalling pathway has an important role in sensing and responding to hyper-osmolar, oxidative and high-temperature stresses in the halophilic fungi H. werneckii. These findings are an important advance in our understanding of the HOG pathway response to stress in H. werneckii, a proposed model organism for studying the salt tolerance of halophilic and halotolerant eukaryotes

Substrate-specific transcription of the enigmatic GH61 family of the pathogenic white-rot fungus Heterobasidion irregulare during growth on lignocellulose

The GH61 represents the most enigmatic Glycoside Hydrolase family (GH) regarding enzymatic activity and importance in cellulose degradation. Heterobasidion irregulare is a necrotizing pathogen and white-rot fungus that causes enormous damages in conifer forests. The genome of H. irregulare allowed identification of ten HiGH61 genes. qRT-PCR analysis separate the HiGH61 members into two groups; one that show up regulation on lignocellulosic substrates (HiGH61A, HiGH61B, HiGH61D, HiGH61G, HiGH61H, and HiGH61I) and a second showing either down-regulation or constitutive expression (HiGH61C, HiGH61E, HiGH61F, and HiGH61J). HiGH61H showed up to 17,000-fold increase on spruce heartwood suggesting a pivotal role in cellulose decomposition during saprotrophic growth. Sequence analysis of these genes reveals that all GH61s except HiGH61G possess the conserved metal-binding motif essential for activity. The sequences also divide into groups having either an insert near the N terminus or an insert near the second catalytic histidine, which may represent extensions of the substrate-binding surface. Three of the HiGH61s encode cellulose-binding modules (CBM1). Interestingly, HiGH61H and HiGH61I having CBM1s are up-regulated on pure cellulose. There was a common substrate-specific induction patterns of the HiGH61s with several reference cellulolytic and hemicellulolytic GHs, this taken together with their low transcript levels on media lacking lignocellulose, reflect the concerted nature of cell wall polymer degradation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-012-4206-x) contains supplementary material, which is available to authorized users

RNA:(guanine-N2) methyltransferases RsmC/RsmD and their homologs revisited – bioinformatic analysis and prediction of the active site based on the uncharacterized Mj0882 protein structure

BACKGROUND: Escherichia coli guanine-N2 (m(2)G) methyltransferases (MTases) RsmC and RsmD modify nucleosides G1207 and G966 of 16S rRNA. They possess a common MTase domain in the C-terminus and a variable region in the N-terminus. Their C-terminal domain is related to the YbiN family of hypothetical MTases, but nothing is known about the structure or function of the N-terminal domain. RESULTS: Using a combination of sequence database searches and fold recognition methods it has been demonstrated that the N-termini of RsmC and RsmD are related to each other and that they represent a "degenerated" version of the C-terminal MTase domain. Novel members of the YbiN family from Archaea and Eukaryota were also indentified. It is inferred that YbiN and both domains of RsmC and RsmD are closely related to a family of putative MTases from Gram-positive bacteria and Archaea, typified by the Mj0882 protein from M. jannaschii (1dus in PDB). Based on the results of sequence analysis and structure prediction, the residues involved in cofactor binding, target recognition and catalysis were identified, and the mechanism of the guanine-N2 methyltransfer reaction was proposed. CONCLUSIONS: Using the known Mj0882 structure, a comprehensive analysis of sequence-structure-function relationships in the family of genuine and putative m(2)G MTases was performed. The results provide novel insight into the mechanism of m(2)G methylation and will serve as a platform for experimental analysis of numerous uncharacterized N-MTases

mRNA:guanine-N7 cap methyltransferases: identification of novel members of the family, evolutionary analysis, homology modeling, and analysis of sequence-structure-function relationships

BACKGROUND: The 5'-terminal cap structure plays an important role in many aspects of mRNA metabolism. Capping enzymes encoded by viruses and pathogenic fungi are attractive targets for specific inhibitors. There is a large body of experimental data on viral and cellular methyltransferases (MTases) that carry out guanine-N7 (cap 0) methylation, including results of extensive mutagenesis. However, a crystal structure is not available and cap 0 MTases are too diverged from other MTases of known structure to allow straightforward homology-based interpretation of these data. RESULTS: We report a 3D model of cap 0 MTase, developed using sequence-to-structure threading and comparative modeling based on coordinates of the glycine N-methyltransferase. Analysis of the predicted structural features in the phylogenetic context of the cap 0 MTase family allows us to rationalize most of the experimental data available and to propose potential binding sites. We identified a case of correlated mutations in the cofactor-binding site of viral MTases that may be important for the rational drug design. Furthermore, database searches and phylogenetic analysis revealed a novel subfamily of hypothetical MTases from plants, distinct from "orthodox" cap 0 MTases. CONCLUSIONS: Computational methods were used to infer the evolutionary relationships and predict the structure of Eukaryotic cap MTase. Identification of novel cap MTase homologs suggests candidates for cloning and biochemical characterization, while the structural model will be useful in designing new experiments to better understand the molecular function of cap MTases