No description available.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus that is closely related to visna virus and more distantly related to the human lentivirus, Human Immunodeficiency Virus type 1 (HIV-1). The CAEV genome contains several small open reading frames (ORFs) that encode viral regulatory proteins. One of these non-structural proteins, Rev-C, is required for cytoplasmic transport of viral un/incompletely spliced mRNAs and efficient viral replication. In HIV-1 and visna virus, Rev is responsible for the temporal shift from non-structural protein synthesis to synthesis of structural proteins that is observed during the viral infectious cycle. Since it encodes a Rev protein, CAEV would be predicted to exhibit a similar temporal shift in gene expression during its replicative cycle. Immunoprecipitation analysis of 35S-pulse labeled, CAEV-infected goat synovial membrane (GSM) cells indicates that Rev-C is more abundant than is Gag at 12 h post-infection (PI); at later times PI Gag predominates. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) experiments using nuclear and cytoplasmic RNA from CAEV-infected GSM cells indicates that the viral unspliced gag mRNA accumulates significantly in the cytoplasm only after Rev is detected. These data indicate that a temporal shift from viral non-structural to structural gene expression occurs in CAEV infected GSM cells.

No description available.

No description available.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Kaposi's sarcoma-associated herpesvirus (KSHV) expresses numerous intronless mRNAs that are unable to access splicing-dependent cellular mRNA nuclear export pathways. To circumvent this problem, KSHV encodes the open reading frame 57 (ORF57) protein, which orchestrates the formation of an export-competent virus ribonucleoprotein particle comprising the nuclear export complex hTREX, but not the exon-junction complex (EJC). Interestingly, EJCs stimulate mRNA translation, which raises the intriguing question of how intronless KSHV transcripts are efficiently translated. Herein, we show that ORF57 associates with components of the 48S pre-initiation complex and co-sediments with the 40S ribosomal subunits. Strikingly, we observed a direct interaction between ORF57 and PYM, a cellular protein that enhances translation by recruiting the 48S pre-initiation complex to newly exported mRNAs, through an interaction with the EJC. Moreover, detailed biochemical analysis suggests that ORF57 recruits PYM to intronless KSHV mRNA and PYM then facilitates the association of ORF57 and the cellular translation machinery. We, therefore, propose a model whereby ORF57 interacts directly with PYM to enhance translation of intronless KSHV transcripts.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 68 pages. Includes Vita. Includes bibliographical references.

The repertoire of cellular functions mediated by Ribonucleic acid (RNA) molecules have expanded considerably during the last two decades. The role played by RNA in controlling and regulating gene expression in viruses, prokaryotes and eukaryotes has been a matter of continuous investigations. This interest has arisen primarily due to the discoveries of cisacting RNA structures like riboswitches, ribosensors and frameshift elements, which are found in either the 5’-, 3’-untranslated regions of mRNA or in the open reading frames. These structures control gene expression at the level of translation by either sequestering the Shine-Dalgarno (SD) sequence to regulate translation initiation or modulating ribosomal positions during an active translation process. Very often, these structures comprise of an RNA pseudoknot and it has been observed that these pseudoknots exist in a dynamic equilibrium with other intermediate structures. This equilibrium could be shifted by several factors including presence of ions, metabolites, temperature and external force. RNA pseudoknots represent the most versatile and ubiquitous class of RNA structures in the cell, whose unique folding topology could be exploited in a number of ways by the cellular machinery. In this thesis, a thorough study of programmed -1 ribosomal frameshifting (-1 PRF) process, which is a well known gene regulation event employed by many RNA viruses, was carried out. -1 PRF is a translation recoding process, necessary for viruses to main-tain a stoichiometric ratio of structural: enzymatic proteins. This ratio varies among different viral species. At the heart of this process, lies an RNA pseudoknot accompanied by a seven nucleotide long sequence motif, which pauses an actively translating ribosome on mRNA and causes it to shift its reading frame. The frameshift inducing efficiency of pseudoknot depends on multiple factors, for example the time scale of ribosomal pause and RNA unfolding, subsequent refolding of structure to native/intermediate states and/or environment conditions. With the aim of illustrating the fundamentals of the process, multiple factors involved in -1 PRF were studied. Chapters 2-4 represent distinct aspects of -1 PRF process, while Chapter 5 discusses a different work concerned with nucleocytoplasmic transport of tRNA carried out by nuclear export receptor Exporting. Chapter 1 gives an overview of the different regulatory activities with which RNA structures and sequences are found to be associated and the evolution of these stud-ies. It discusses the different types of structural motifs found to constitute tertiary RNA structure and secondary structure prediction and determination techniques. A brief description of ab initio RNA structure modeling and other relevant tools and methodologies used in this work has been presented. Details of techniques used in each study have been provided in relevant chapters. Chapter 2 describes how local factors like ionic conditions, hydration patterns, presence of protonated residues and single residue mutations affect the structural dynamics of an RNA pseudoknot involved in -1 PRF from a plant luteovirus. Single residue mutations in the loop regions or certain base-pair inversions in the stem regions of pseudoknot increase the frameshift inducing ability of the pseudoknot structure, while some others decrease this efficiency. However, it was not clear how the changes made to the wild-type (WT) RNA pseudoknot from Beet Western Yellow Mosaic virus were affecting the global structure in terms of its dynamics and other parameters. To study this, multiple all-atom molecular dynamics simulations (MD) were performed on WT and mutant structures created in silico. The effect of presence and absence of magnesium ions on the structural geometry was also studied. The analysis was done to identify the increase/decrease in the number of hydrogen bonds formed by Watson-Crick base-pairs in stem region or non Watson-Crick pairs between stem and loop. Ionic and water densities were analyzed and the role of potential ribosome-pseudoknot interaction was elaborated. With the aim of mimicking ribosome induced unfolding of an RNA pseudoknot, steered molecular dynamics pulling experiments were performed. This work was done primarily to understand the unfolding pathway of Hairpin(H)-type pseudoknots in general and the intermediate structures formed. Chapter 3 describes the thermodynamics and mechanics associated with the mechanical pulling of -1 PRF inducing RNA pseudoknot and its mutants described in previous chapter. Analysis of the trajectories reveal relative unfolding patterns in terms of disruption of various hydrogen bonds. This study allowed us to pinpoint the kind of intermediate structures being formed during pulling and whether these intermediate structures correspond to any known secondary structures, such as simple stem-loops. This information could be used for gaining insights into the folding pathways of these structures. An RNA pseudoknot stimulates -1 PRF in conjunction with a heptanucleotide “slippery site” and an intervening spacer sequence. A comprehensive study of analyzing the sequence signatures and composition of all overlapping gene segments harboring these frameshift elements from four different RNA virus families was carried out. Chapter 4 describes the sequence composition of all overlapping gene segments in Astroviridae, Coronaviridae, Retroviridae and Luteoviridae viral families which are known to employ -1 PRF process for maintaining their protein products. Sequence analysis revealed preference for GC bases in the structure forming sequence regions. A comparative study between multiple sequence alignment and secondary structure prediction revealed that while pseudoknots have a clear preference for specific base-pairs in their stem regions, viral families that employ a hairpin loop as -1 PRF structure, doesn’t show this preference. Information derived from secondary structure prediction was then used for RNA ab initio modeling to generate tertiary structures. Furthermore, the structural parameters were calculated for the helices of the frameshift inducing pseudoknots and were compared with the values calculated for a set of non -1 PRF inducing H-type pseudo-knots. This study highlighted the differences between -1 PRF pseudoknots and other H-type pseudoknot structures as well as specific sequence and structural preferences of the former. Chapter 5 discusses the dynamics of a tRNA transport factor Exportint (Xpot), which transports mature tRNA molecules from nucleus to cytoplasm and belongs to Importitβ family of proteins. The global conformational dynamics of other transport receptors has been reported earlier, using coarse-grained modeling and Elastic Network Models (ENMs), but a detailed description of the dynamics at an all-atomic resolution was lacking. This transport requires association of Xpot with RanGTP, a G-protein, in the nucleus and hydrolysis of RanGTP in the cytoplasm. The chain of events leading to tRNA release from Xpot after RanGTP hydrolysis was not studied previously. With these objectives, several molecular complexes containing Xpot bound to Ran or tRNA or both in the GTP and GDP ligand states as well as free Xpot structures in nuclear and cytosolic forms were studied. A combination of conventional and accelerated molecular dynamics simulations was used to study these molecular complexes. The study highlighted various aspects associated with tRNA release and conformational change which occurs in Xpot in cytosolic form. The nuclear to cytosolic state transition in Xpot could be attributed to large fluctuations in C-terminal region and dynamic hinge-points located between specific HEAT repeats. A secondary role of Xpot in controlling the quality of tRNA transport has been proposed based on multiple sequence and structure alignment with Importin-β protein. The loss of critical contacts like hydrogen bonds and salt bridges between Xpot/Ran and Xpot/tRNA interface was evaluated in order to study the initial effects of RanGTP hydrolysis and how it influences receptor-cargo binding. This study revealed various aspects of tRNA transport process by Xpot, not understood previously. The results presented in this thesis illustrate the role of RNA sequence elements and pseudoknots present in RNA viruses in modulating -1 PRF process and how multiple environmental factors affect -1 PRF inducing ability of the structure. From the studies of Xpot and its complexes, the effects of GTP hydrolysis leading to tRNA dissociation have been presented and the progression of conformational transition in Xpot after tRNA dissociation has been highlighted. Chapter 6 summarizes major conclusions of this thesis work. The refolding of single stranded RNA chains, subjected to a previous unfolding simulation is studied. Appendix A describes this work and initial results. Appendix B describes the effect of improved molecular dynamics force fields, containing corrections for χ torsion angle for RNA, on the conformation of tertiary RNA structures. Part of the work presented in this thesis has been reported in the following publications. 1.Asmita Gupta and Manju Bansal. Local Structural and Environmental Factors De-fine the Efficiency of an RNA Pseudoknot Involved in Programmed Ribosomal Frameshift Process. J. Phys. Chem. B. 118 (41), pp 11905-11920. 2014 2.Asmita Gupta, Senthilkumar Kailasam and Manju Bansal. Insights Into Nucleo-cytoplasmic Transport of tRNA by Exportin-t. Manuscript under review. List of manuscripts that are being prepared from the work reported in Chapter 3 in this thesis. 1 Asmita Gupta and Manju Bansal. The role of sequence effects on altering the un-folding pathway of an RNA pseudoknot: a steered molecular dynamics study. Manuscript in preparation. 2 Asmita Gupta and Manju Bansal. Molecular basis for nucleocytoplasmic transport of tRNA by Exportin-t. Journal of Biomolecular Structure and Dynamics, May;33 Suppl 1:59-60, 2015

No description available.

Small interfering RNAs (siRNAs), microRNAs (miRNAs), and piRNAs (piRNA) are conserved classes of small single-stranded ~21-30 nucleotide (nt) RNA guides that repress eukaryotic gene expression using distinct RNA Induced Silencing Complexes (RISCs). At its core, RISC is composed of a single-stranded small RNA guide bound to a member of the Argonaute protein family, which together bind and repress complementary target RNA. miRNAs target protein coding mRNAs—a function essential for normal development and broadly involved in pathways of human disease; small interfering RNAs (siRNA) defend against viruses, but can also be engineered to direct experimental or therapeutic gene silencing; piwi associated RNAs (piRNAs) protect germline genomes from expansion of parasitic nucleic acids such as transposons. Using the fruit fly, Drosophila melanogaster, as a model organism we seek to understand how small silencing RNAs are made and how they function. In Drosophila, miRNAs and siRNAs are proposed to have parallel, but separate biogenesis and effector machinery. miRNA duplexes are excised from imperfectly paired hairpin precursors by Dicer1 and loaded into Ago1; siRNA duplexes are hewn from perfectly paired long dsRNA by Dicer2 and loaded into Ago2. Contrary to this model we found one miRNA, miR-277, is made by Dicer1, but partitions between Ago1 and Ago2 RISCs. These two RISCs are functionally distinct—Ago2 could silence a perfectly paired target, but not a centrally bulged target; Ago1 could silence a bulged target, but not a perfect target. This was surprising since both Ago1 and Ago2 have endonucleolytic cleavage activity necessary for perfect target cleavage in vitro. Our detailed kinetic studies suggested why—Ago2 is a robust multiple turnover enzyme, but Ago1 is not. Along with a complementary in vitro study our data supports a duplex sorting mechanism in which Diced duplexes are released, and rebind to Ago1 or Ago2 loading machinery, regardless of which Dicer produced them. This allows structural information embedded in small RNA duplexes to direct small RNA loading into Ago1 and/or Ago2, resulting in distinct regulatory outputs. Small RNA sorting also has chemical consequences for the small RNA guide. Although siRNAs were presumed to have the signature 2′, 3′ hydroxyl ends left by Dicer, we found that small RNAs loaded into Ago2 or Piwi proteins, but not Ago1, are modified at their 3´ ends by the RNA 2´-O-methyltransferase DmHen1. In plants Hen1 modifies the 3´ ends all small RNAs duplexs, protecting and stabilizing them. Implying a similar function in flies, piRNAs are smaller, less abundant, and their function is perturbed in hen1 mutants. But unlike plants, small RNAs are modified as single-strands in RISC rather than as duplexes. This nicely explains why the dsRNA binding domain in plant Hen1 was discarded in animals, and why both dsRNA derived siRNAs and ssRNA derived piRNAs are modified. The recent discovery that both piRNAs and siRNAs target transposons links terminal modification and transposon silencing, suggesting that it is specialized for this purpose.