PACCMIT/PACCMIT-CDS: identifying microRNA targets in 3' UTRs and coding sequences.

The purpose of the proposed web server, publicly available at http://paccmit.epfl.ch, is to provide a user-friendly interface to two algorithms for predicting messenger RNA (mRNA) molecules regulated by microRNAs: (i) PACCMIT (Prediction of ACcessible and/or Conserved MIcroRNA Targets), which identifies primarily mRNA transcripts targeted in their 3' untranslated regions (3' UTRs), and (ii) PACCMIT-CDS, designed to find mRNAs targeted within their coding sequences (CDSs). While PACCMIT belongs among the accurate algorithms for predicting conserved microRNA targets in the 3' UTRs, the main contribution of the web server is 2-fold: PACCMIT provides an accurate tool for predicting targets also of weakly conserved or non-conserved microRNAs, whereas PACCMIT-CDS addresses the lack of similar portals adapted specifically for targets in CDS. The web server asks the user for microRNAs and mRNAs to be analyzed, accesses the precomputed P-values for all microRNA-mRNA pairs from a database for all mRNAs and microRNAs in a given species, ranks the predicted microRNA-mRNA pairs, evaluates their significance according to the false discovery rate and finally displays the predictions in a tabular form. The results are also available for download in several standard formats.

Searching the coding region for microRNA targets.

Finding microRNA targets in the coding region is difficult due to the overwhelming signal encoding the amino acid sequence. Here, we introduce an algorithm (called PACCMIT-CDS) that finds potential microRNA targets within coding sequences by searching for conserved motifs that are complementary to the microRNA seed region and also overrepresented in comparison with a background model preserving both codon usage and amino acid sequence. Precision and sensitivity of PACCMIT-CDS are evaluated using PAR-CLIP and proteomics data sets. Thanks to the properly constructed background, the new algorithm achieves a lower rate of false positives and better ranking of predictions than do currently available algorithms, which were designed to find microRNA targets within 3' UTRs.

Analysis of the accessibility of CLIP bound sites reveals that nucleation of the miRNA:mRNA pairing occurs preferentially at the 3'-end of the seed match.

To find out whether the AGO-miRNA complex is more sensitive to the accessibility of a particular region inside the seed match, we analyze in detail the accessibility of a wide set of miRNA binding sites validated by PAR-CLIP and HITS-CLIP experiments. Our analysis reveals that nucleotides at the 3'-end of bound seed matches are significantly more accessible than nucleotides at the 5'-end as well as nucleotides at any positions in the unbound seed matches. We show that the accessibility of a single nucleotide at the 3'-end is more effective than the accessibility of several nucleotides at the 5'-end in discriminating between functional and nonfunctional binding sites. Analysis of mRNA and protein fold changes induced by miRNA overexpression demonstrates that genes with accessible nucleation regions at the 3'-end are down-regulated more strongly than genes whose accessible nucleation regions are located elsewhere within the seed match. We also observed an increase in the precision of the miRNA target prediction algorithm PACMIT when accessibility toward the 3'-end of the seed match was required. The pronounced sensitivity of the AGO-miRNA complex to the accessibility of the 3'-end of the seed match suggests that, in most cases, nucleation occurs in this region. We show that this conclusion is consistent with previous experimental studies.

Optimal use of conservation and accessibility filters in microRNA target prediction.

It is generally accepted that filtering microRNA (miRNA) target predictions by conservation or by accessibility can reduce the false discovery rate. However, these two strategies are usually not exploited in a combined and flexible manner. Here, we introduce PACCMIT, a flexible method that filters miRNA binding sites by their conservation, accessibility, or both. The improvement in performance obtained with each of these three filters is demonstrated on the prediction of targets for both i) highly and ii) weakly conserved miRNAs, i.e., in two scenarios in which the miRNA-target interactions are subjected to different evolutionary pressures. We show that in the first scenario conservation is a better filter than accessibility (as both sensitivity and precision are higher among the top predictions) and that the combined filter improves performance of PACCMIT even further. In the second scenario, on the other hand, the accessibility filter performs better than both the conservation and combined filters, suggesting that the site conservation is not equally effective in rejecting false positive predictions for all miRNAs. Regarding the quality of the ranking criterion proposed by Robins and Press and used in PACCMIT, it is shown that top ranking interactions correspond to more downregulated proteins than do the lower ranking interactions. Comparison with several other target prediction algorithms shows that the ranking of predictions provided by PACCMIT is at least as good as the ranking generated by other conservation-based methods and considerably better than the energy-based ranking used in other accessibility-based methods.

Efficient use of accessibility in microRNA target prediction.

Considering accessibility of the 3'UTR is believed to increase the precision of microRNA target predictions. We show that, contrary to common belief, ranking by the hybridization energy or by the sum of the opening and hybridization energies, used in currently available algorithms, is not an efficient way to rank predictions. Instead, we describe an algorithm which also considers only the accessible binding sites but which ranks predictions according to over-representation. When compared with experimentally validated and refuted targets in the fruit fly and human, our algorithm shows a remarkable improvement in precision while significantly reducing the computational cost in comparison with other free energy based methods. In the human genome, our algorithm has at least twice higher precision than other methods with their default parameters. In the fruit fly, we find five times more validated targets among the top 500 predictions than other methods with their default parameters. Furthermore, using a common statistical framework we demonstrate explicitly the advantages of using the canonical ensemble instead of using the minimum free energy structure alone. We also find that 'naïve' global folding sometimes outperforms the local folding approach.

Characterization of activity landscapes using 2D and 3D similarity methods: consensus activity cliffs.

Activity landscape characterization has been demonstrated to be a valuable tool in lead optimization, virtual screening, and computational modeling of active compounds. In this work, we present a general protocol to explore systematically the activity landscape of a lead series using 11 2D and 3D structural representations. As a test case we employed a set of 48 bicyclic guanidines (BCGs) with kappa-opioid receptor binding affinity, identified in our group. MACCS keys, graph-based three point pharmacophores, circular fingerprints, ROCS shape descriptors, and the TARIS approach, that compares structures based on molecular electrostatic potentials, were employed as orthogonal descriptors. Based on 'activity cliffs' common to a series of descriptors, we introduce the concept of consensus activity cliffs. Results for the current test case suggest that the presence or absence of a methoxybenzyl group may lead to different modes of binding for the active BCGs with the kappa-opioid receptor. The most active compound (IC50 = 37 nM) is involved in a number of consensus cliffs making it a more challenge query for future virtual screening than would be expected from affinity alone. Results also reveal the importance of screening high density combinatorial libraries, especially in the "cliff-rich" regions of activity landscapes. The protocol presented here can be applied to other data sets to develop a consensus model of the activity landscape.

Radial scan of the molecular electrostatic potential of RNA double helices: an application to the enzyme-tRNA recognition.

We introduced a method to characterize quantitatively the molecular electrostatic potential (MEP) of the minor and major grooves of base pairs located at nucleic acid double helices. By means of a radial MEP scan, we obtained a n-tuple of potential values corresponding to each groove, which can be analyzed by plotting the MEP values as a function of the angle in the radial scan. We studied base pairs of two different tRNAs, relevant in the recognition process with their cognate aminoacyl tRNA synthetases (aaRSs), in order to correlate their electrostatic behavior with the corresponding aminoacylation activity. We analyzed the first three base pairs of the Escherichia coli tRNA(Ala) acceptor stem, finding several cases where the MEP profiles obtained from the plots are in agreement with the reported aminoacylation activities. Additionally, a non-hierarchical clustering performed over the MEP n-tuples resulted in meaningful classifications that correlate with the activity and with the predicted stereochemistry of the reaction. We also studied the first two base pairs of the E. coli tRNA(Thr) acceptor stem but constraining the analysis to the angle intervals that seem relevant for the binding sites of the enzyme. These intervals were deduced from the ThrRS-tRNA(Thr) complex crystal structure. In this case, we also found a good agreement between the MEP profiles and the activity, supporting the idea that the tRNA identity elements function is to allow an optimal electrostatic complementarity between the aminoacyl-tRNA synthetase and the tRNA.

CARACTERIZACIÓN DEL TALLO ACEPTOR DEL tARN MEDIANTE DESCRIPTORES LOCALES BASADOS EN CARGAS PARCIALES / tRNA ACCEPTOR STEM CHARACTERIZATION BY MEANS OF LOCAL DESCRIPTORS BASED ON PARTIAL CHARGES / CARACTERIZAÇÃO DO TALO ACEITADOR DO tRNA MEDIANTE DESCRITORES LOCAIS BASEADOS EM CARGAS PARCIAIS

En este trabajo se caracteriza la distribución de carga del tallo aceptor del tARN, considerando todas las posibles combinaciones de pares Watson-Crick. El estudio se realizó con 256 fragmentos moleculares de 10 nucleótidos que modelan los tres primeros pares del tallo aceptor, la base diferenciadora y el extremo CCA. Para caracterizar los nucleótidos se proponen dos descriptores locales basados en la distribución de carga de la base nitrogenada de cada nucleótido, los cuales se calculan a partir de las cargas parciales de Mulliken obtenidas de cálculos HF/6-31G. La caracterización y clasificación de los tallos según estos descriptores mostró cómo la base diferenciadora tiene un comportamiento particular respecto a los demás nucleótidos del tallo y una fuerte influencia sobre el extremo CCA. La clasificación de nueve variaciones del tallo aceptor del tARNAla mostró una buena relación estructura-actividad que pone en evidencia la bondad de los descriptores propuestos para caracterizar de manera local la distribución de carga de estas biomoléculas.

In this work the charge distribution of the tRNA acceptor stem is characterized, considering all the possible Watson- Crick base pair combinations. 256 RNA fragments modeled by 10 nucleotides were used in order to model the first three pairs of the acceptor stem, the discriminator base and the CCA end. We propose two local charge descriptors based on the charge distribution of the nitrogenated base to characterize each nucleotide. These descriptors were computed from atomic partial charges derived from HF/6-31G calculations. From the characterization and classification of the stems according to the proposed descriptors, we found a special behavior for the discriminator base (in contrast to the other positions) and a strong effect of this position on the CCA end. The classification of nine variations of the tRNAAla acceptor stem showed a good structure-activity relationship that makes evident the usefulness of the proposed descriptors to characterize the local charge distributions of these biomolecules.

Nesse estudo é caracterizada a distribuição da carga do talo aceitador considerando- se todas as combinações possíveis dos pares Watson-Crick. O estudo realizouse com 256 fragmentos moleculares dos 10 nucleotídeos que modelam os três primeiros pares do talo aceitador, a base diferenciadora e o extremo CCA. Com o intuito de caracterizar cada nucleotídeo, foram propostos dois descritores locais baseados na distribuição de carga da base nitrogenada de cada nucleotídeo, os quais se calculam a partir das cargas parciais de Mulliken obtidas de cálculos HF/6-31G. A caracterização e classificação dos talos segundo esses descritores demonstrou um particular comportamento da base diferenciadora em relação aos demais nucleotídeos do talo e uma forte influência sobre o extremo CCA. A classificação de nove variações do talo aceitador do tRNA mostrou uma boa relação estrutura-atividade que colocam em evidência a utilidade dos descritores propostos para caracterizar de maneira local a distribuição de carga dessas bio-moléculas.

Graph theoretical similarity approach to compare molecular electrostatic potentials.

In this work we introduce a graph theoretical method to compare MEPs, which is independent of molecular alignment. It is based on the edit distance of weighted rooted trees, which encode the geometrical and topological information of Negative Molecular Isopotential Surfaces. A meaningful chemical classification of a set of 46 molecules with different functional groups was achieved. Structure--activity relationships for the corticosteroid binding affinity (CBG) of 31 steroids by means of hierarchical clustering resulted in a clear partitioning in high, intermediate, and low activity groups, whereas the results from quantitative structure--activity relationships, obtained from a partial least-squares analysis, showed comparable or better cross-validated correlation coefficients than the ones reported for previous methods based solely in the MEP.