Recent Research Highlights
Development of a computational method for template-free RNA 3D structure modeling and folding simulations
Researchers from the Bujnicki laboratory developed SimRNA: a new method for computational RNA 3D structure prediction, which uses a coarse-grained representation, relies on the Monte Carlo method for sampling the conformational space, and employs a statistical potential to approximate the energy and identify conformations that correspond to biologically relevant structures. SimRNA can fold RNA molecules using only sequence information, and, on established test sequences, it recapitulates secondary structure with high accuracy, including correct prediction of pseudoknots. For modeling of complex 3D structures, it can use additional restraints, derived from experimental or computational analyses, including information about secondary structure and/or long-range contacts. SimRNA also can be used to analyze conformational landscapes and identify potential alternative structures. SimRNA is available as a standalone program for the Linux and MacOSX operating systems at http://genesilico.pl/simrna/ and it is free for non-commercial use by academic users. An article describing the SimRNA method has been published in Nucleic Acids Res. 2015 doi: 10.1093/nar/gkv1479 [epub 2015 Dec 19]
Data from MODOMICS have been included in the RNAcentral database
Data from the MODOMICS database of RNA modification pathways [http://modomics.genesilico.pl/], developed by the Bujnicki group with the help of several collaborating groups, have been included in the fourth release of RNAcentral database. RNAcentral [http://rnacentral.org] is a comprehensive database of accessioned ncRNA sequences that integrates information from an international consortium. The most recent release of RNAcentral features seven new Expert Databases (dictyBase, Greengenes, LNCipedia, MODOMICS, NONCODE, PomBase, and SILVA) and modified nucleotides from PDB and MODOMICS. More details can be found on the RNAcentral blog [http://blog.rnacentral.org/2015/11/rnacentral-release-4.html]. The main contributor to this development on behalf of the Bujnicki group has been Magdalena Mika.
Development of a computational method for experimental determination of RNA and DNA crystal structures
The number of experimentally determined structures of nucleic acid molecules, including nucleic acid-protein complexes, is increasing rapidly, in line with recent discoveries and growing interest in biological functions exerted by nucleic acids, beyond their protein-coding capacity. In general the method-of-choice for studies of macromolecular structures is X-ray crystallography. However, nucleic acids crystallography, unlike the protein crystallography, still lacks sufficient methodology to facilitate a straightforward crystal structure determination process. In particular, computational tools that automatically build a crystal structure model into an experimental electron-density map are markedly less developed for nucleic acids than for proteins.
Researchers from the Bujnicki laboratory developed BrickworX, a computer program that builds crystal structure models of nucleic acid molecules using recurrent motifs, including double-stranded helices. It builds on the RNA Bricks database (http://iimcb.genesilico.pl/rnabricks) previously developed in the Bujnicki group, which stores information about recurrent RNA 3D motifs and their interactions that are found in experimentally determined RNA structures and RNA-protein complexes. The advantage of Brickworx compared to other available methods, is that it can process poor-quality electron density maps, in which only a fraction of nucleotide residues can be modeled with confidence. For instance if only a fraction of the phosphate group positions can be detected, a correctly placed complete motif matching these phosphates can be built into the electron-density map. In the first step, Brickworx searches for electron-density peaks that may correspond to the phosphate groups, it may also take into account phosphate group positions provided by the user. Subsequently, based on comparison of the phosphorus atom 3D patterns with the database of nucleic acid fragments, it finds matching positions of double stranded helical motifs (A-RNA or B-DNA) in the unit cell. If the target structure is RNA, the helical fragments are further extended with recurrent RNA motifs from a fragment library that contains single-stranded segments. Finally, the matched motifs are merged and refined in real-space to find most likely conformations, including the fit of sequence to the electron density map.
The Brickworx program is available for download and as a web server at http://iimcb.genesilico.pl/brickworx . The article describing the Brickworx method and its implementation have been published in Acta Crystallogr D Biol Crystallogr. 2015 Mar;71(Pt 3):697-705. [epub 2015 Feb 26]
Identification and characterization of a nuclease that cleaves dsRNA sequence-specifically
Molecular biology has made tremendous progress in the 20th century owing to the discovery of restriction endonucleases that cleave double stranded DNA molecules in specific sequences. However, so far no “restriction enzymes for double stranded RNA” have been found in nature or engineered in the laboratory, thus limiting the progress of RNA research. The availability of enzymes that are able to cleave double stranded RNA in a sequence-dependent manner could potentially facilitate the development of new nucleic acid manipulation techniques.
Researchers from the Bujnicki laboratory discovered and characterized the ability of a member of the RNase III superfamily from Bacillus subtilis, called 'BsMiniIII', to cleave double stranded RNA in a sequence-dependent manner. The experimental analysis has been prompted by a bioinformatics analysis that identified a certain position in the structure of RNase III as a potential site for insertions that could cause the enzyme to make sequence-specific contacts to the RNA. BsMiniIII was found to possess a natural variant of such an insertion, compared to sequence-non-specific RNase III enzymes. The Bujnicki group analyzed sites cleaved by the BsMiniIII enzyme, in a limited digest of bacteriophage Φ6 dsRNA, and identified a nucleotide sequence that was cleaved with high preference. They defined nucleotide residues within that sequence that affected the efficiency of the cleavage. They have also determined a crystal structure of the BsMiniIII enzyme and constructed a model of the protein-RNA complex. The insertion that distinguishes MiniIII enzymes from other members of RNase III family is structurally disordered in the absence of the RNA, and is essential for the specific cleavage, but not for dsRNA binding. Altogether, these results suggest that BsMiniIII may serve as a prototype of a sequence-specific dsRNase that could possibly be developed towards a “restriction enzyme for RNA”.
The findings were published in Nucleic Acids Research in a report that was awarded a status of a Breakthrough Article, on January 9, 2014. Breakthrough articles at NAR describe studies that solve a long-standing problem in their field, or provide exceptional new insight and understanding into an area of research that will clearly motivate and guide new research opportunities and directions. They represent the top papers that NAR receives for publication, and are selected by the Editors based on nominations by authors and/or reviewers, and on the subsequent recommendation of the reviewers and editorial board members.
Determination of structure and mechanism of action of human methyltransferases that act on the cap structure in RNA
Collaborating scientists at International Institute of Molecular and Cell Biology in Warsaw (IIMCB) and at University of Warsaw (UW) have determined spatial structures and the mechanism of action of proteins called methyltransferases, which are responsible for the enzymatic formation of mature mRNA molecules in human cells. This discovery illuminated the process by which the human methyltransferases recognize and chemically modify the so-called cap structure at one end of mRNA. This cap structure is essential for the synthesis of proteins in the cells. The team led by Bujnicki found that human methyltransferases recognize the cap in mRNA differently from proteins used by viruses to perform a similar reaction, thus providing a framework for targeting these viral proteins by drugs that are safe for humans. Using X-ray crystallography (spearheaded by graduate student Mirosław Śmietański, co-supervised by Bujnicki and Nowotny), the team at IIMCB was able to determine atomic details of a complex formed by an enzymatically active catalytic domain of human cap methyltransferase 1 (CMTr1) with a short ‘capped’ RNA. Scientists have had great difficulty obtaining the protein-RNA complex in a form that is suitable for the imaging of its structure by crystallography. For example a suitable RNA molecule was difficult to obtain and it was chemically synthesized by the Darżynkiewicz group at UW. Researchers in the Bujnicki group used the crystallographically determined structure as a template to model computationally an analogous complex formed by human cap methyltransferase 2 (CMTr1) with an RNA that was a product of a reaction made by CMTr1. They also performed biochemical experiments on CMTr1 and CMTr2 (led by Maria Werner, a postdoctoral researcher at the Bujnicki group) to confirm the importance of individual amino acid residues predicted to be involved in the recognition of RNA based on the crystallographic and computational models of molecular structures.
The findings are published in an article in Nature Communications, on January 9, 2014.
Successful development of an enzyme that sequence-specifically cuts RNA in RNA/DNA hybrids
Ribonucleases (RNases) are valuable tools applied in the analysis of RNA sequence, structure and function. Their substrate specificity is limited to recognition of single bases or distinct secondary structures in the substrate. Currently, there are no RNases available for purely sequence-dependent fragmentation of RNA. The researchers from the Bujnicki laboratory, in collaboration with Dr. Nowotny (head of Laboratory of Protein Structure in IIMCB) have developed a new enzyme that cleaves the RNA strand in DNA-RNA hybrids 5 nucleotides from a specific recognition sequence. The design invoved the combination of computational structure prediction with experimental analyses. The engineered enzyme is a fusion of two functionally distinct domains: a RNase HI, that hydrolyzes RNA in DNA-RNA hybrids in processive and sequence-independent manner, and a zinc finger that recognizes a sequence in DNA-RNA hybrids. Methods for engineering zinc finger domains with new sequence specificities are readily available, making it feasible to acquire a library of RNases that recognize and cleave a variety of sequences, much like the commercially available assortment of restriction enzymes. Potentially, zinc finger-RNase HI fusions may, in addition to in vitro applications, be used in vivo for targeted RNA degradation. The results of this research are subject of a patent application and have been accepted for publication in Nucleic Acids Res.
Computational structural analysis of the mRNA-splicing machinery in human cells
The spliceosome is one of the largest molecular machines known. It performs the excision of introns from eukaryotic pre-mRNAs. In human cells it comprises five RNAs, over one hundred “core” proteins and more than one hundred additional associated proteins. The details of the spliceosome mechanism of action are unclear, because only a small fraction of spliceosomal proteins have been characterized structurally in high resolution. Researchers from the Bujnicki laboratory have carried out a comprehensive analysis of the human spliceosomal proteome. They discovered that almost a half of the combined sequence of proteins abundant in the spliceosome is predicted to be intrinsically disordered, at least when the individual proteins are considered in isolation. They have also correlated the type and abundance of disorder with different protein functions. For regions without an experimental structure in the ordered part of the spliceosomal proteome, they predicted and modeled three-dimentional structures. They have also developed a database of structural models for the entire spliceosomal proteome, called SpliProt3D. The results of this work enable multiscale modeling of the structure and dynamics of the entire spliceosome and its subcomplexes and will guide further research toward understanding of the molecular mechanism of mRNA splicing. The results of this research have been published in Nucleic Acids Res. 2012 Aug 1;40(15):7046-65 [Epub 2012 May 9] and PLoS Comput Biol. 2012 Aug;8(8):e1002641 [Epub 2012 Aug 9].
Successful engineering of a restriction enzyme substrate preference aided by protein structure prediction
Researchers from the Bujnicki laboratory have succesfully altered a sequence specificity of restriction enzyme R.MwoI by a single amino acid substitution, based on a structural model of the enzyme generated in the absence of a crystal structure. The results of the study prove the utility of protein modeling as a tool to guide protein engineering. This work has successfully demonstrated that a single residue replacement can lead to the development of new, different sequence preferences. This finding illustrates the potential of restriction enzymes to develop new sequence specificities in the nature. Together with the new procedure for engineering of thermophilic restriction enzymes, this study provides a toolkit for rational engineering of sequence specificities in Type II restriction enzymes. The results of this research have been accepted for publication as a Featured Article in Nucleic Acids Research 2012 June 25 doi:10.1093/nar/gks570 and artwork illustrating this article (author: Małgorzata Durawa) has been selected for a cover image of the Nucleic Acids Research issue contaiing this article.
The MetaDisorder meta-server is available
Researchers from the Bujnicki laboratory have developed a seriws of protein disorder/order predictors, which excelled e.g. in the CASP benchmarks. These methods have now been made available via the MetaDisorder web portal and the article describing their development and tests has been published in Bioinformatics 2012 May 24;13(1):111.
ModeRNA is available as a web server
Researchers from the Bujnicki laboratory have made ModeRNA available as a freely available web server. Research published in Bioinformatics 2011 Sep 1;27(17):2441-2.
The GeneSilico metaserver for protein disorder prediction shows excellent performance in CASP9 Rankings of the 9th Community-Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP9) organized by the Protein Structure Prediction Center have shown high positions of methods developed in the Bujnicki laboratory. In particular, the updated version of GeneSilico metaserver for protein disorder prediction, has been again ranked as number 1 in its category. High scores have been also obtained by our new experimental methods for protein modeling by recombination of fragments (RecombineIt) and model quality assessment tools.
The Bujnicki team performs well in RNA Puzzles
Rankings of RNA Puzzles, the first "CASP-like" experiment for RNA 3D structure modeling organized by the team of Eric Westof at the University of Strasbourg have shown very good performance of tools for RNA modeling developed in the Bujnicki laboratory, including ModeRNA (a method for comparative modeling, see below) and SimRNA (a method for template-free modeling, unpublished). Research published in the RNA journal, 2012 Apr;18(4):610-25
REPAIRtoire: a database of DNA repair pathways
Researchers from the Bujnicki laboratory, in collaboration with Joanna Krwawicz (IBB PAS, Warsaw), have developed REPAIRtoire, a database of processes and molecular entities involved in the repair of damaged DNA in the cells. It contains information about chemical structures of damaged nucleotides, biochemical pathways leading from a particular type of altered DNA to repaired DNA, genes and enzymes responsible for the biochemical reactions, and diseases caused by defects in DNA repair systems. REPAIRtoire connects DNA damage to potential causes of each lesion as well as to effects if they are not removed. It provides tools with which to study important aspects of DNA metabolism in the cell. REPAIRtoire has been published in the Nucleic Acids Research annual database issue, 2011 Jan;39:D788-92.
ModeRNA: a new method for comparative modeling of RNA 3D structures
Researchers from the Bujnicki laboratory have developed ModeRNA, a new method for template-based modeling of RNA 3D structures. A special feature of ModeRNA is the ability to model posttranscriptional modifications.Research published in Nucleic Acids Research 2011 May 1;39(10):4007-22.
GeneSilico metaserver tops the CASP8 ranking in protein disorder prediction
The GeneSilico human predictors' group and several servers developed in the Bujnicki laboratory achieved high positions in rankings of the 8th Community-Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP8), organized by the Protein Structure Prediction Center. Servers for protein structure prediction and model quality assessment achieved respectable positions in their respective categories. The most successful was the GeneSilico metaserver for protein disorder prediction, which ranked as number 1 in its category. These successful servers are freely available to all academic researchers via the GeneSilico toolkit.
Characterization of ubiquitin-associated (UBA) domain in inhibitor of apoptosis (IAP) proteins.
Janusz Bujnicki contributed a structural model to a collaborative study (led by Pascal Meier from the Breakthrough Toby Robins Breast Cancer Research Centre in London) that has characterized an evolutionarily conserved ubiquitin-associated (UBA) domain in inhibitor of apoptosis (IAP) proteins. An article entitled "IAPs contain an evolutionarily conserved ubiquitin-binding domain that regulates NF-kappaB as well as cell survival and oncogenesis" has been published in Nature Cell Biology and describes the following findings: UBA domain enables IAPs to bind to Lys 63-linked polyubiquitin and is essential for the oncogenic potential of cIAP1, to maintain endothelial cell survival and to protect cells from TNF-alpha-induced apoptosis. Moreover, the UBA domain is required for XIAP and cIAP2-MALT1 to activate NF-kappaB. The UBA domain of cIAP2-MALT1 stimulates NF-kappaB signalling by binding to polyubiquitylated NEMO. This publication identified IAPs as ubiquitin-binding proteins that contribute to ubiquitin-mediated cell survival, NF-kappaB signalling and oncogenesis.
New rRNA modification enzymes discovered and characterized
Researchers from the Bujnicki laboratory predicted (with bioinformatics) and then experimentally confirmed that two so far uncharacterized open reading frames encode methyltransferases acting on 23S rRNA. YbeA is the m3Psi methyltransferase RlmH that targets nucleotide 1915, while YccW is the m5C methyltransferase RlmI specific for nucleotide 1962. The experimental analysis was done in collaboration with a group in Odense headed by prof.Stephen Douthwaite (University of Southern Dennmark),
Moreover, researchers from the Bujnicki laboratory analyzed the crystal structure of YccW/RlmI solved by a collaborating group (prof. Jayaraman Sivaraman, University of Singapore) and found that it provides a missing link in the evolutionary history of several different families of methyltransferases that modfy cytosine in DNA or RNA as well as uridine in RNA. They have also identified a new putative RNA-binding domain dubbed 'EEHEE'. This domain is common to RlmI and several other RNA-modification enzymes, including methyltransferases involved in m5U formation and in Wye base biosynthesis. The results of this research have been published in RNA (2008 Oct;14(10):2234-44 - one article about RlmH) and in Journal of Molecular Biology (two articles about RlmI structure and functional analysis)
R.PabI restriction enzyme reveals a new protein fold and other unusual features
Researchers from the Bujnicki laboratory participated in the discovery of a protein with a new three-dimensional fold. This finding was a result of a collaboration with a group of Japanese researchers headed by prof. Ichizo Kobayashi (University of Tokyo). R.PabI is a sequence-specific nuclease that cuts DNA within GATC sequences. It belongs to Type II restriction enzymes, proteins that are used as commercial reagents to 'cut and paste' DNA fragments in the laboratory.
R.PabI is unusual (and very valuable) for three reasons: First, R.PabI cuts the DNA to produce a 3'-TA "sticky end" that is unique among restriction enzymes, therefore it can be used for a novel way of 'pasting' of cleaved DNA molecules. Second, unlike nearly all restriction enzymes characterized to date, R.PabI does not require metal ions for its nuclease activity. Third, R.PabI exhibits a new three-dimensional architecture, a feature that is very interesting from the point of view of evolutionary biology and protein folding. Researchers from the Bujnicki laboratory predicted that R.PabI may exhibit a new type of structure (research published in Nucleic Acids Research, 2005 Jul 21; Vol. 33, No. 13: e112) and collaborated with prof. Kobayashi and his co-workers to analyze the R.PabI structure solved by X-ray crystallography. The structure of R.PabI and its analysis has been published in Nucleic Acids Research 2007;35(6):1908-18.
Polish researchers excel in protein structure prediction
Two groups from the Bujnicki laboratory achieved very high scores in the 6th Community-Wide Experiment on the Critical Assessment of Techniques for Protein Structure Prediction (CASP-6), organized by the Protein Structure Prediction Center. According to the official rankings for individual categories presented by the expert assessors at the CASP-6 meeting in Gaeta (Italy), the "Kolinski-Bujnicki" group (a duumvirate of Janusz Bujnicki and Andrzej Kolinski from the University of Warsaw) was ranked 1st/2nd (depending on the scoring system) in the New Folds category and 3rd in the Comparative Modeling category. The "GeneSilico" group of students from the Bujnicki lab was ranked 2nd in the Fold-Recognition (Homology) category. Assessors' presentations and detailed evaluations of individual models are available on-line at the CASP-6 website.
In the unofficial ranking compiled by Dr. Jeffrey Skolnick, based on a fully automated assessment of all CASP-6 models regardless of their difficulty, "Kolinski-Bujnicki" and "GeneSilico" ranked 2nd and 5th, respectively. These scores are similar to those obtained in the previous edition of the experiment (CASP-5, 2002), where "Bujnicki-Janusz" and "GeneSilico" ranked 2nd and 5th in the unofficial overall ranking compiled by Dr. Michael Levitt, with the 1st position in Comparative Modeling for "Bujnicki-Janusz".