Functionally distinct nucleic acid binding sites for a group I intron encoded RNA maturase/DNA homing endonuclease

A large number of group I introns encode a family of homologous proteins that either promote intron splicing (maturases) or are site-specific DNA endonucleases that function in intron mobility (a process called "homing"). Genetic studies have shown that some of these proteins have both activities, yet how a single protein carries out both functions remains obscure. The similarity between respective DNA-binding sites and the RNA structure near the 5' and 3' splice sites has fueled speculation that such proteins may use analogous interactions to perform both functions. The Aspergillus nidulans mitochondrial COB group I intron encodes a bi-functional protein, I-AniI, that has both RNA maturase and site-specific DNA endonuclease activities in vitro. Here, we show that I-AniI shows distinctive features of the endonuclease family to which it belongs, including highly specific, tight binding and sequential DNA strand cleavage. Competition experiments demonstrate that I-AniI binds the COB intron RNA even in saturating concentrations of its DNA target site substrate, suggesting that the protein has a separate binding site for RNA. In addition, we provide evidence that two different DNA-binding site mutants of I-AniI have little effect on the protein's RNA maturation activity. Since RNA splicing is likely a secondary adaptation of the protein, these observations support a model in which homing endonucleases may have developed maturase function by utilizing a hitherto "non-functional" protein surface.     

A novel mechanism for protein-assisted group I intron splicing

Previously it was shown that the Aspergillus nidulans (A.n.) mitochondrial COB intron maturase, I-AniI, facilitates splicing of the COB intron in vitro. In this study, we apply kinetic analysis of binding and splicing along with RNA deletion analysis to gain insight into the mechanism of I-AniI facilitated splicing. Our results are consistent with I-AniI and A.n. COB pre-RNA forming a specific but labile encounter complex that is resolved into the native, splicing-competent complex. Significantly, kinetic analysis of splicing shows that the resolution step is rate limiting for splicing. RNA deletion studies show that I-AniI requires most of the A.n. COB intron for binding suggesting that the integrity of the I-AniI-binding site depends on overall RNA tertiary structure. These results, taken together with the observation that A.n. COB intron lacks significant stable tertiary structure in the absence of protein, support a model in which I-AniI preassociates with an unfolded COB intron via a "labile" interaction that facilitates correct folding of the intron catalytic core, perhaps by resolving misfolded RNAs or narrowing the number of conformations sampled by the intron during its search for native structure. The active intron conformation is then "locked in" by specific binding of I-Anil to its intron interaction site.     

Multiple sequence alignment with arbitrary gap costs: computing an optimal solution using polyhedral combinatorics

Multiple sequence alignment is one of the dominant problems in computational molecular biology. Numerous scoring functions and methods have been proposed, most of which result in NP-hard problems. In this paper we propose for the first time a general formulation for multiple alignment with arbitrary gap-costs based on an integer linear program (ILP). In addition we describe a branch-and-cut algorithm to effectively solve the ILP to optimality. We evaluate the performances of our approach in terms of running time and quality of the alignments using the BAliBase database of reference alignments. The results show that our implementation ranks amongst the best programs developed so far.     

RNA: versatility in form and function

RNA performs a remarkable range of functions in all cells. In addition to its central role in information transfer from DNA to protein, it is essential for functions as diverse as RNA processing, chromosome end-maintenance and dosage compensation. The versatility of RNA derives from its unique ability to use direct readout via base-pairing for sequence specific targeting (or templating) in combination with its capacity to form elaborate three dimensional structures. Such structures can perform catalysis or serve as protein recognition surfaces. In this short review, we attempt to give a flavor for the diversity of functional RNAs in the cell and highlight, using selected examples, two quite distinct activities, catalysis and sequence specific targeting. Within each section, we discuss how the lessons we have learned from these systems may apply to other, less well understood, RNAs.     

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.     

Follow my eyes: the gaze of politicians reflexively captures the gaze of ingroup voters

Studies in human and non-human primates indicate that basic socio-cognitive operations are inherently linked to the power of gaze in capturing reflexively the attention of an observer. Although monkey studies indicate that the automatic tendency to follow the gaze of a conspecific is modulated by the leader-follower social status, evidence for such effects in humans is meager. Here, we used a gaze following paradigm where the directional gaze of right- or left-wing Italian political characters could influence the oculomotor behavior of ingroup or outgroup voters. We show that the gaze of Berlusconi, the right-wing leader currently dominating the Italian political landscape, potentiates and inhibits gaze following behavior in ingroup and outgroup voters, respectively. Importantly, the higher the perceived similarity in personality traits between voters and Berlusconi, the stronger the gaze interference effect. Thus, higher-order social variables such as political leadership and affiliation prepotently affect reflexive shifts of attention.     

A shared RNA-binding site in the Pet54 protein is required for translational activation and group I intron splicing in yeast mitochondria

The Pet54p protein is an archetypical example of a dual functioning (‘moonlighting’) protein: it is required for translational activation of the COX3 mRNA and splicing of the aI5β group I intron in the COX1 pre-mRNA in Saccharomyces cerevisiae mitochondria (mt). Genetic and biochemical analyses in yeast are consistent with Pet54p forming a complex with other translational activators that, in an unknown way, associates with the 5′ untranslated leader (UTL) of COX3 mRNA. Likewise, genetic analysis suggests that Pet54p along with another distinct set of proteins facilitate splicing of the aI5β intron, but the function of Pet54 is, also, obscure. In particular, it remains unknown whether Pet54p is a primary RNA-binding protein that specifically recognizes the 5′ UTL and intron RNAs or whether its functional specificity is governed in other ways. Using recombinant protein, we show that Pet54p binds with high specificity and affinity to the aI5β intron and facilitates exon ligation in vitro. In addition, Pet54p binds with similar affinity to the COX3 5′ UTL RNA. Competition experiments show that the COX3 5′UTL and aI5β intron RNAs bind to the same or overlapping surface on Pet54p. Delineation of the Pet54p-binding sites by RNA deletions and RNase footprinting show that Pet54p binds across a similar length sequence in both RNAs. Alignment of the sequences shows significant (56%) similarity and overlap between the binding sites. Given that its role in splicing is likely an acquired function, these data support a model in which Pet54p's splicing function may have resulted from a fortuitous association with the aI5β intron. This association may have lead to the selection of Pet54p variants that increased the efficiency of aI5β splicing and provided a possible means to coregulate COX1 and COX3 expression.     

Structure based virtual screening of ligands to identify cysteinyl leukotriene receptor 1 antagonist

Montelukast and Zafirlukast are known leukotriene receptor antagonists prescribed in asthma treatment. However, these fall short as mono therapy and are frequently used in combination with inhaled glucocorticosteroids with or without long acting beta 2 agonists. Therefore, it is of interest to apply ligand and structure based virtual screening strategies to identify compounds akin to lead compounds Montelukast and Zafirlukast. Hence, compounds with structures having 95% similarity to these compounds were retrieved from NCBI׳s PubChem database. Compounds similar to lead were grouped and docked at the antagonist binding site of cysteinyl leukotriene receptor 1. This exercise identified compounds UNII 70RV86E50Q (Pub Cid 71587778) and Sure CN 9587085 (Pub Cid 19793614) with higher predicted binding compared to Montelukast and Zafirlukast. It is shown that the compound Sure CN 9587085 showed appreciable ligand receptor interaction compared to UNII 70RV86E50Q. Thus, the compound Sure CN 9587085 is selected as a potent antagonist to cysteinyl leukotriene receptor 1 for further consideration in vitro and in vivo validation.     

1001 optimal PDB structure alignments: integer programming methods for finding the maximum contact map overlap.

Protein structure comparison is a fundamental problem for structural genomics, with applications to drug design, fold prediction, protein clustering, and evolutionary studies. Despite its importance, there are very few rigorous methods and widely accepted similarity measures known for this problem. In this paper we describe the last few years of developments on the study of an emerging measure, the contact map overlap (CMO), for protein structure comparison. A contact map is a list of pairs of residues which lie in three-dimensional proximity in the protein's native fold. Although this measure is in principle computationally hard to optimize, we show how it can in fact be computed with great accuracy for related proteins by integer linear programming techniques. These methods have the advantage of providing certificates of near-optimality by means of upper bounds to the optimal alignment value. We also illustrate effective heuristics, such as local search and genetic algorithms. We were able to obtain for the first time optimal alignments for large similar proteins (about 1,000 residues and 2,000 contacts) and used the CMO measure to cluster proteins in families. The clusters obtained were compared to SCOP classification in order to validate the measure. Extensive computational experiments showed that alignments which are off by at most 10% from the optimal value can be computed in a short time. Further experiments showed how this measure reacts to the choice of the threshold defining a contact and how to choose this threshold in a sensible way.     

An allosteric-feedback mechanism for protein-assisted group I intron splicing

The I-AniI maturase facilitates self-splicing of a mitochondrial group I intron in Aspergillus nidulans. Binding occurs in at least two steps: first, a specific but labile encounter complex rapidly forms and then this intermediate is slowly resolved into a native, catalytically active RNA/protein complex. Here we probe the structure of the RNA throughout the assembly pathway. Although inherently unstable, the intron core, when bound by I-AniI, undergoes rapid folding to a near-native state in the encounter complex. The next transition includes the slow destabilization and docking into the core of the peripheral stacked helix that contains the 5' splice site. Mutational analyses confirm that both transitions are important for native complex formation. We propose that protein-driven destabilization and docking of the peripheral stacked helix lead to subtle changes in the I-AniI binding site that facilitate native complex formation. These results support an allosteric-feedback mechanism of RNA-protein recognition in which proteins engaged in an intermediate complex can influence RNA structure far from their binding sites. The linkage of these changes to stable binding ensures that the protein and RNA do not get sequestered in nonfunctional complexes.