-Computational Detection of Abundant Long-range Nucleotide Co-variation in Genomes- A novel computational methodology was developed to detect long-range co-variation on a genomic scale producing high and medium confidence interactinos networks. To test the algorithm, it was applied to a set of aligned drosophila genomes where it was found that there are prevalent long-range co-variations between exons of mRNAs and non-coding RNAs, including endo-siRNAs. Most of the co-variations appear to be the result of synchronized mutations, not reverse complementarity. The genes found to co-vary are enriched for functions related to regionalization as well as neural and developmental processes. In a lower confidence interaction network there is a bias towards genes that have cellular component annotations such as plasma membrane or cytoskeleton. This may be an indicator of a widespread phenomenon of hitchhiking, in which transcripts bind to other RNAs that are part of ribonucleoprotein complexes which are transported along the cytoskeleton. These interactions may occur in a statistical size dependent manner. These results indicate that RNA-RNA long-range interactions are a widespread phenomenon that is important to a variety of cellular processes. - Discovery and Characterization of a Novel Translational Enhancers - 3'UTRs of cellular and viral mRNAs harbor elements that function in gene expression by enhancing translation using unknown mechanisms. To determine the function of these elements we previously used the Turnip crinkle virus (TCV). TCV is translated in a cap-independent fashion and contains a 3'region that together with the 5'UTR synergistically enhances translation. We recently discovered another element of this type in the Pea enation mosaic virus, however, this translational enhancer element acts in a somewhat different way. We used a set of computational tools, including RNA2D3D for 3D RNA modeling, developed in our lab, and experimental methods to show that this element is T-shaped, which is reminiscent of TCV's similarity to tRNA. We showed that it binds ribosomes and engages in a long range kissing loop RNA-RNA interaction. Its functionality suggests a means for RNA 5'/3'cyclization and thus a mechanism for translational enhancement. It appears that the existence of these novel elements is suggesting alternate translational mechanisms that may be found in eukaryotic organisms. - Understanding the Structure and Function of the Dengue Virus - It has recently been reported that there are over 400 million cases of dengue fever in the world, 4 times more than previously reported with 10% leading to severe forms of the disease. Using our massively parallel genetic algorithm for RNA folding we showed that the core region of the 3'untranslated region of dengue virus RNA can form 2 dumbell structures of unequal frequencies of occurrence. It was experimentally shown that structural motifs formed from these dumbells are important for viral replication. Also it was shown that there is a cooperative synergy with both dumbells for translation. Thus, the cis-acting elements in the core region of dengue virus are required for both replication and optimal translation. Recently, we performed a detailed structural analysis of the dengue minigenome using acylation techniques (SHAPE), lead induced hydrolysis and site directed mutagenesis. Results indicate protein independent interactions between the 5'and 3'terminal regions corroborating many of the predicted interactions and shedding light on the pseudoknot interactions found in the 3'UTR and the sequence dependency on the 5'cyclization region. -The Role of Ions and Flanking Bases in the Dimerization of HIV-1- Experimentally it has been shown that the characterization of HIV-1 kissing loop formation differs depending on the subtype of the virus. It has been shown that subtype-B monomers dimerize at high salt concentrations or in the presence of magnesium ions while subtype-A monomers will only dimerize with a magnesium ion bound to the flanking G273 phosphate group or the phosphate group of G274 regardless of the salt concentration. We found using computer simulations that at low concentrations both types of monomer hairpin loops were significantly deformed and the bases in the hairpin loop that are associated with dimerization were turned inward. At high salt concentrations the subtype-B monomer maintained a shape conducive to dimerization, while subtype-A still showed significant deformations. Also the flanking bases in subtype-B helped to stabilize the conformation while the flanking base G273 in subtype-A caused deformation. However, when magnesium ions were present and bound to the G273 or G274 phosphate groups, base G273 maintained a conformation that stabilized the loop for dimerization. These results are important for understanding the mechanisms that are involved in the HIV-1 virus life cycle. -Classifying Pre-miRNA via Combinatorial Feature Mining and Boosting MicroRNAs are non-coding RNAs consisting of about 22 nucleotides that are derived from precursor molecules. These precursors usually fold into stem-loop hairpin structures. When scanning genomes it is difficult to distinguish false positive pre-miRNAs from the real thing. In this research a new method was developed for identifying and classifying pre-miRNAs. A combinatorial feature mining approach was used to discover a good set of features. These feature sets were then used to train support vector machines to obtain classification models. A boosting algorithm was then applied to further enhance the accuracy. Results indicate significant improvement over previous methods. -Discovering Common Folding Patterns in two RNA Sequences- An efficient dynamic programming algorithm was developed that uses ordered labeled trees for discovering the largest common RNA substructures given 2 RNA sequences. This algorithm can also be used to discover repeated regions of RNA secondary structure. -Multiscale Modeling of Double Helical DNA and RNA Unified through Lie Groups- In order to further understand motions that are inherent in nucleic acid helices, several means of mechanical modeling, continuous and discrete, of double-stranded nucleic acid helical structures (DNA and RNA)were reviewed and a new analysis method using Lie groups was used to reconcile the models. Rigid body motions were analyzed. At the most coarse level worm-like chains were used with anisotropic bending stiffness. It was then shown that bi-rod models converge to this for long enough lengths. We then showed how full atomic resolution molecular dynamics and experimental results obtained from atomic force microscopy relate to the models.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Kasprzak, Wojciech K; Shapiro, Bruce A (2014) MPGAfold in dengue secondary structure prediction. Methods Mol Biol 1138:199-224
Tamim, Saleh; Vo, Dat T; Uren, Philip J et al. (2014) Genomic analyses reveal broad impact of miR-137 on genes associated with malignant transformation and neuronal differentiation in glioblastoma cells. PLoS One 9:e85591
Kim, Taejin; Shapiro, Bruce A (2013) The role of salt concentration and magnesium binding in HIV-1 subtype-A and subtype-B kissing loop monomer structures. J Biomol Struct Dyn 31:495-510
Bindewald, Eckart; Shapiro, Bruce A (2013) Computational detection of abundant long-range nucleotide covariation in Drosophila genomes. RNA 19:1171-82
Zhong, Ling; Wang, Jason T L; Wen, Dongrong et al. (2013) Effective classification of microRNA precursors using feature mining and AdaBoost algorithms. OMICS 17:486-93
Wang, Jinbu; Zuo, Xiaobing; Yu, Ping et al. (2009) A method for helical RNA global structure determination in solution using small-angle x-ray scattering and NMR measurements. J Mol Biol 393:717-34
Santhanam, Arti N; Bindewald, Eckart; Rajasekhar, Vinagolu K et al. (2009) Role of 3'UTRs in the translation of mRNAs regulated by oncogenic eIF4E--a computational inference. PLoS One 4:e4868
de Sousa Abreu, Raquel; Sanchez-Diaz, Patricia C; Vogel, Christine et al. (2009) Genomic analyses of musashi1 downstream targets show a strong association with cancer-related processes. J Biol Chem 284:12125-35
Stupina, Vera A; Meskauskas, Arturas; McCormack, John C et al. (2008) The 3'proximal translational enhancer of Turnip crinkle virus binds to 60S ribosomal subunits. RNA 14:2379-93
Martinez, Hugo M; Maizel Jr, Jacob V; Shapiro, Bruce A (2008) RNA2D3D: a program for generating, viewing, and comparing 3-dimensional models of RNA. J Biomol Struct Dyn 25:669-83