Discovery and Characterization of a Novel Translational Enhancers3'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 act 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 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.Characteristics that Determine Abundance of Two-Thirds of Proteins in a Human Cell LineTranscription, mRNA decay, translation, and protein degradation all contribute to steady state protein concentrations in multi-cellular eukaryotes. In this research, experimental and computational studies were done to determine the absolute protein and mRNA abundances in cellular lysates from the human Daoy medulloblastoma cell line, and the properties that contributed to these abundances. Sequence features related to translation and protein degradation explained two-thirds of protein abundance variation. mRNA sequence lengths, amino acid properties, upstream open reading frames and secondary structures in the 5'untranslated region (UTR) showed the strongest individual correlations for protein concentrations. In a combined model, characteristics of the coding region and the 3'UTR explained a larger proportion of protein abundance variation than characteristics of the 5'UTR.Cis Acting Elements in the 3'UTR of Dengue VirusOver 50 million case of dengue fever are reported each year 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.Correlating SHAPE Signatures with 3D RNA StructuresSelective 2-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is a relatively easy technique for the quantitative analysis of RNA secondary structure. Low SHAPE signal values are correlated with Watson-Crick base pairing, and high values indicate positions that are single-stranded within the RNA structure. The relationship of the measured SHAPE signal to structural properties such as non-Watson-Crick base pairing has thus far not been thoroughly investigated. In this research we present results of SHAPE experiments performed on a set of seven RNAs with published 3D structures. We found that the RNA SHAPE signal depends on the type of base pairs a nucleotide is involved in, its ability to stack and its context. This is important for improving computational/experimental methods for RNA structure prediction.The Role of Ions and Flanking Bases in the Dimerization of HIV-1Experimentally 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 BoostingMicroRNAs 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.Data Mining of Functional RNA Structures in Genomic SequencesThe normal functions of genomes depend on the precise expression of mRNAs and ncRNAs, such as microRNAs. These ncRNAs and functional RNA structures (FRSs) act as regulators or response elements for cellular factors, participate in transcription, post-transcriptional processing, and translation. In RNA-based regulation, the regulatory RNAs are often correlated with distinct higher-order structures. Computational simulations indicated that a large number of FRSs are significantly more structured and thermodynamically stable. Various computational tools have been developed and the structural features of ncRNAs and FRSs have been determined. We report our efforts in the computational discovery of structured features of ncRNAs and FRSs within complex genomes.Discovering Common Folding Patterns in two RNA SequencesAn 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.Characterizing Structural Features for Small Regulatory RNAs in E. ColiSmall regulatory RNAs are highly abundant noncoding RNAs (ncRNA) found in bacterial genomes. These small regulatory ncRNAs (sRNAs) can regulate the synthesis of proteins by mediating mRNA transcription, translation and stability. Furthermore, they also control the activity of specific proteins by binding to them. In this research, we describe a general computational approach for identifying the distinct structure of sRNAs in the Escherichia coli (E. coli) genomes by a quantitative measure that is the energy difference between the optimal structure folded from a sequence segment and its corresponding optimal restrained structure where all base pairings formed in the original optimal structure are excluded. Our results indicated that most of the known small ncRNAs in E. coli K12 have high statistical structural significance and are thermodynamically stable.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC008382-29
Application #
8552600
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
29
Fiscal Year
2012
Total Cost
$451,557
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Shapiro, Bruce A; Le Grice, Stuart F J (2016) Advances in RNA structure determination. Methods 103:1-3
Song, Yang; Hua, Lei; Shapiro, Bruce A et al. (2015) Effective alignment of RNA pseudoknot structures using partition function posterior log-odds scores. BMC Bioinformatics 16:39
Belew, Ashton Trey; Meskauskas, Arturas; Musalgaonkar, Sharmishtha et al. (2014) Ribosomal frameshifting in the CCR5 mRNA is regulated by miRNAs and the NMD pathway. Nature 512:265-9
Gao, Feng; Kasprzak, Wojciech K; Szarko, Christine et al. (2014) The 3' untranslated region of Pea Enation Mosaic Virus contains two T-shaped, ribosome-binding, cap-independent translation enhancers. J Virol 88:11696-712
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
Sztuba-Solinska, Joanna; Teramoto, Tadahisa; Rausch, Jason W et al. (2013) Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome. Nucleic Acids Res 41:5075-89

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