Plant luteoviral RNA viruses employ -1 frameshifting for the production of P1 and P1-P2 fusion proteins important for viral replication. Luteoviral pseudoknots are characterized by three adenosines in the 3' side of loop L2 known to be important for maintaining frameshifting efficiency and pseudoknot stability. A proposed P1-P2 mRNA pseudoknot from sugarcane yellow leaf virus (ScYLV) was of interest since it contained two adenosine to cytidine substitutions in L2. Functional analysis shows that the in vitro frameshifting efficiency is greater (~15%) than any other luteoviral pseudoknot. The NMR-derived solution structure of the ScYLV RNA pseudoknot shows that C25 is looped out of the triplex structure and the 3' most L2 cytidine (C27) and A24 form cis Watson-Crick/sugar-edge interactions with C14 and C15 in stem S1, respectively. Thus, the ScYLV pseudoknot maintains a similar triple helical architecture as other luteoviral pseudoknots. Surprisingly, the frameshifting efficiency of the C27A ScYLV pseudoknot is decreased by ~8 fold relative to wild-type ScYLV. The solution structure of the C27A ScYLV RNA exhibits a global fold similar to the wild-type RNA; however, distinct hydrogen bonding interactions at the helical junction are observed. Specifically, C8+ in the C8+ major groove base triple moves ~2.3 relative to the accepting (G12-C28) base pair relative to the WT RNA. New NMR experiments have been developed and/or applied to confirm Watson-Crick base pairs and tertiary structural interactions in the PEMV-1 and ScYLV pseudoknots by direct observation of trans hydrogen bond scalar couplings. In addition, intrabase couplings in cytidine and adenosine have been measured, providing a valuable tool for the assignment of amino and N3/N1 resonances in RNA. Finally, thermodynamic analysis of the pairwise coupling between the major groove and minor groove tertiary structural hydrogen bonds at the helical junction have been investigated by monitoring the thermal unfolding of WT, dC14, C27A, and dC14/C27A RNAs as a function of pH. Favorable pairwise coupling characterized the WT ScYLV and BWYV RNAs, while unfavorable coupling characterized the poorly functional C27A ScYLV RNA. The implications of these structural, functional, and thermodynamic findings on the mechanism of frameshift stimulation is discussed.

Our understanding of the functions played by RNA molecules is expanded with the understanding of RNA structures. Except for primary structure, RNA molecules present pairings within a sequence, which is called RNA secondary structure. Since its discovery, RNA secondary structure has drawn considerable attention because it is widely appeared. Many programs for RNA secondary structure prediction have been developed, including [4, 20, 38, 39, 46]. Based on our knowledge, however, there is a family of RNA secondary structure which can not be covered by any of these algorithms. And even without considering this family, none of programs can cover all other structures in Rfam data-set. These structures are found to be important in many biological processes, for example, chromosome maintenance, RNA processing, protein biosynthesis. And efficient structure prediction can give direction for experimental investigations. Here, we present a general algorithm with a new grammar: Vertical Tree Grammar (VTG) which has stochastic context-free grammar architecture for RNA secondary structure prediction. VTG significantly expands the class of structures that can be handled, including all structures that can be covered by other paper, and all structures in Rfam data-set. Our algorithm runs in O(n^6) time, and it's precision is reasonable high, with average sensitivity and specificity over 70%. / published_or_final_version / Computer Science / Master / Master of Philosophy

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Mobile group II introns are retroelements that are found in prokaryotes, archaea, and the organelles of plants and fungi, but not in the nuclear genomes of eukaryotes. They consist of a catalytically active RNA and intron-encoded reverse transcriptase, which together promote site-specific integration into DNA sites in a mechanism called retrohoming. The group II intron Ll.LtrB has been developed into a programmable, DNA-targeting agent called "targetron", which is widely used in bacteria and an attractive technology for gene targeting in eukaryotes. However, group II intron genome targeting in human cells has not been equivocally shown. This dissertation focuses on the hypothesis that the low Mg2+-concentrations found in higher eukaryotes present a natural barrier to group II introns. First, I studied E. coli host proteins that aid group II intron retrohoming and found that synthesis of a second DNA-strand relies on host replication restart proteins. Next, I demonstrated that mutations in the distal stem of the catalytic core domain V (DV) improve Ll.LtrB retrohoming in a low Mg2+-concentration E. coli mutant and in biochemical assays. These results suggest that DV is involved in an RNA-folding step that becomes rate limiting at low Mg2+. Subsequently, I performed directed evolution of the intron RNA by injecting in vitro prepared mutant intron libraries into Xenopus laevis oocyte nuclei. The mutations were analyzed using Roche 454 sequencing to generate an intron fitness landscape, which revealed conserved positions and potentially beneficial mutations, enabling enhanced retrohoming in Xenopus oocytes. Finally, I used a hybrid Pol II/T7 Ll.LtrB eukaryotic expression system to show that high exogenous MgCl2 in the growth media enables retrohoming into plasmids and genomic DNA in human cells. In vivo directed evolution and mutation analyses using PacBio RS circular consensus sequencing indicated that only a few mutations may improve intron activity in human cells. This dissertation provides evidence that efficient group II intron retrohoming in human cells is limited by low Mg2+-concentrations and develops new approaches for overcoming this limitation to enable use of group II introns for gene targeting in higher organisms. / text

No description available.

Research about ribonucleic acid (RNA) is gaining popularity as we widen our knowledge about its function. But to properly examine new structures, we need robust computational tools to analyse different properties. One of such tools is structural superposition, which is a method to align two structures over each other and quantify their similarity. This tool can be used on tertiary RNA structures for visual comparison, clustering or the assessment of their function. The aim of this thesis is to present a novel approach for achieving RNA superposition using information about secondary RNA structure and its link to trees. Tree edit distance algorithms are used to compare the trees, and a multitude of methods for generating the structural superposition from the calculated tree similarities is presented. The new method is aligned in the context of existing works, and its accuracy is compared to the best current approaches for structural superposition. The implementation can be accessed at https://github.com/gyfis/rawted.

It has long been recognized that our understanding of how RNA adapts its complex three-dimensional structure and undergoes conformational fluctuations has played a central role in our understanding of the biological functions of RNA. Our current understanding of the vast and diverse set of RNA conformational dynamics is the culmination of several decades of biophysical research applying several ensemble and single-molecule techniques. In this journey, each of the biophysical techniques have provided a unique perspective into the dynamic processes of RNA and revealed information about distinct RNA dynamics occurring over a broad range of timescales. In recent years, a new, promising single-molecule biophysical technique called single-molecule field effect transistors (smFETs) has been developed. Because smFETs do not rely on fluorophore reporters of conformation or mechanical (un)folding forces, they provide a unique approach that enables single-molecule studies of RNA conformational dynamics observed at microsecond temporal resolution for a long period of time. The broad range of timescales opens immediate prospects for smFETs to provide a unique perspective into understanding RNA conformational dynamics that are presently inaccessible in other single-molecule approaches. The primary focus of this thesis is to understand how RNA stem-loops undergo folding and unfolding. Stem-loops are one of the most common secondary structural motifs in RNA and act as a fundamental building block for complex RNA structures. Despite their fundamental importance, a complete unifying picture of the folding mechanism of RNA stem-loops has been difficult to achieve, primarily due to the rugged nature of their folding energy landscapes. In Chapter 2, experimental methods that were developed to enable smFET studies of RNA conformational dynamics are described. This includes the development of a high-throughput fabrication process that generates high signal to noise ratio (SNR) smFET devices and the development and validation of nucleic acid tethering strategies that enables controlled tethering of biomolecules onto smFET devices. Utilizing these methods, Chapter 3 establishes smFET as a general single-molecule approach to characterize the folding dynamics of RNA stem-loops. Finally, Chapter 4 explores the use of smFETs to investigate the molecular mechanism in which a model RNA stem-loop undergoes folding and unfolding. Collectively, this thesis demonstrates how smFETs can be applied to uniquely capture and describe the folding energy landscapes of RNA and reveal new insights to how RNAs fold and unfold.

No description available.

Inhibition of protein synthesis serves as a general measure of cellular consequences of chemical stress. A few proteins are translated selectively and influence cell fate. How these proteins can bypass the general control of translation remains unknown. We found that low to mild doses of oxidants induce de novo translation of the NRF2 protein. Here we demonstrate the presence of a G-quadruplex structure in the 5' untranslated region (UTR) of NRF2 mRNA, as measured by circular dichroism, nuclear magnetic resonance, and dimethylsulfate footprinting analyses. Such a structure is important for 5'-UTR activity, since its removal by sequence mutation eliminated H2O2-induced activation of the NRF2 5' UTR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics revealed elongation factor 1 alpha (EF1a) as a protein binding to the G-quadruplex sequence. Cells responded to H2O2 treatment by increasing the EF1a protein association with NRF2 mRNA, as measured by RNA-protein interaction assays. The EF1a interaction with small and large subunits of ribosomes did not appear to change due to H2O2 treatment, nor did post translational modifications, as measured by two-dimensional (2-D) Western blot analysis. Since NRF2 encodes a transcription factor essential for protection against tissue injury, our data have revealed a novel mechanism of cellular defense involving de novo NRF2 protein translation governed by the EF1a interaction with the G-quadruplex in the NRF2 5' UTR during oxidative stress.

Myotonic dystrophy (DM) is one of the most common forms of muscular dystrophy, characterized by its hallmark symptom myotonia. DM is an autosomal dominant disease caused by a toxic gain of function RNA. The toxic RNA is produced from expanded non-coding CTG/CCTG repeats, and these CUG/CCUG repeats sequester a family of RNA binding proteins. The Muscleblind-like (MBNL) family of RNA binding proteins are sequestered to the expanded CUG/CCUG repeats. The MBNL proteins are regulators of alternative splicing, and their sequestration to the toxic RNA leads to mis-splicing events, which are believed to cause the symptoms observed in DM patients. A previously reported screen for small molecules used to identify compounds that could disrupt MBNL from binding the toxic CUG repeats found that pentamidine was able to rescue splicing defects associated with DM. Herein, we present a new class of molecules (phenolsulphonphthaleins) that inhibited MBNL1/CUG repeat complex formation in a competitive electrophoretic mobility shift assay (EMSA). Additionally, one of these molecules, bromophenol blue (BPB), acted in a synergistic manner with the previously described inhibitor pentamidine. We also demonstrated that the halogenation of the phenolsulphonphthalein dyes is an important factor for activity. Moreover, we presentant analysis of a series of methylene linker variants of pentamidine that revealed heptamidine (an analog of pentamidine) could reverse splicing defects in a DM1 tissue culture model and rescue myotonia in a DM1 mouse model. Finally, we report on a new crystal structure of CUG repeats, crystallized in the context of a GAAA tetraloop/receptor which facilitated ordered packing within the crystal. This structure was consistent with previous structures showing that the repeats are essentially A-form RNA, despite having a U-U mismatch every third base pair. We also identified six types of U-U mismatch in the context of the 5'CUG/3'GUC motif, suggesting that the interactions between the uridines are dynamic. This structure also contains the highest resolution GAAA tetraloop/receptor structure (1.95 Å) reported to date. This dissertation includes previously unpublished co-authored material.