Biochemistry, Biophysics, and Structural Biology Commons^™

Chemical Damage To Mrna And Its Impact On Ribosome Quality-Control And Stress-Response Pathways In Eukaryotic Cells, Liewei Yan

Arts & Sciences Electronic Theses and Dissertations

Ribosome often faces defective adducts that disrupt its movement along the mRNA template. These adducts are primarily caused by chemical damage to mRNA and are highly detrimental to the decoding process on the ribosome. Hence, unless dealt with, chemical damage to RNA has been hypothesized to lead to the production of toxic protein products. Even more detrimental is the ability of damaged mRNA to drastically affect ribosome homeostasis through stalling. This in turn would lead to greatly diminished translation capacity of cells. Therefore, the inability of cells to recognize and resolve translational-stalling events is detrimental to proteostasis and could even …

Launching The Next Generation : Transcriptional Regulation During Oogenesis, Alicia K. Mccarthy

Legacy Theses & Dissertations (2009 - 2024)

Germ cells give rise to gametes and link generations by passing genetic information from parent to offspring. Gametes arise from, in many sexually reproducing organisms, germline stem cells (GSCs) which are set aside early during development. GSCs have an amazing capacity to undergo self-renewal to give rise to a pool of undifferentiated cells, while also differentiating to generate specialized germ cells such as haploid gametes. Upon female GSC differentiation, mitotically dividing germ cells can initiate meiosis, and mature within a follicle. During maturation, the specified oocyte is provided with a trust fund of RNAs and proteins for the next generation …

Iron-Dependent Cleavage Of Ribosomal Rna During Oxidative Stress In The Yeast Saccharomyces Cerevisiae, Jessica A Zinskie, Arnab Ghosh, Brandon M Trainor, Daniel Shedlovskiy, Dimitri G Pestov, Natalia Shcherbik

Rowan-Virtua School of Osteopathic Medicine Faculty Scholarship

Stress-induced strand breaks in rRNA have been observed in many organisms, but the mechanisms by which they originate are not well-understood. Here we show that a chemical rather than an enzymatic mechanism initiates rRNA cleavages during oxidative stress in yeast (Saccharomyces cerevisiae). We used cells lacking the mitochondrial glutaredoxin Grx5 to demonstrate that oxidant-induced cleavage formation in 25S rRNA correlates with intracellular iron levels. Sequestering free iron by chemical or genetic means decreased the extent of rRNA degradation and relieved the hypersensitivity of grx5Δ cells to the oxidants. Importantly, subjecting purified ribosomes to an in vitro iron/ascorbate …

Regulation Of Gene Expression By Rna Binding Proteins And Micrornas, Kyle Cottrell

Arts & Sciences Electronic Theses and Dissertations

Regulation of gene expression is essential to life. Post-transcriptional regulation of gene expression is a complex process with many inputs that lead to changes in localization, translation and stability of mRNAs. The translation and stability of many mRNAs is regulated by cis-elements, such as mRNA-structure or codon optimality; and by trans-acting factors such as RBPs and miRNAs. Here I report on the complex interactions between RBPs, miRNAs and characteristics of their target mRNAs in respect to effects on translation and RNA stability.

Using a reporter based approach we studied modulation of microRNA-mediated repression by various mRNA characteristics. We observed the …

One-Step Hot Formamide Extraction Of Rna From Saccharomyces Cerevisiae, Daniel Shedlovskiy, Natalia Shcherbik, Dimitri G Pestov

Rowan-Virtua School of Osteopathic Medicine Faculty Scholarship

Current methods for isolating RNA from budding yeast require lengthy and laborious steps such as freezing and heating with phenol, homogenization with glass beads, or enzymatic digestion of the cell wall. Here, extraction with a solution of formamide and EDTA was adapted to isolate RNA from whole yeast cells through a rapid and easily scalable procedure that does not require mechanical cell lysis, phenol, or enzymes. RNA extracted with formamide-EDTA can be directly loaded on gels for electrophoretic analysis without alcohol precipitation. A simplified protocol for downstream DNase treatment and reverse transcription reaction is also included. The formamide-EDTA extraction of …

Endonucleolytic Cleavage In The Expansion Segment 7 Of 25s Rrna Is An Early Marker Of Low-Level Oxidative Stress In Yeast, Daniel Shedlovskiy, Jessica A Zinskie, Ethan Gardner, Dimitri G Pestov, Natalia Shcherbik

Rowan-Virtua School of Osteopathic Medicine Faculty Scholarship

The ability to detect and respond to oxidative stress is crucial to the survival of living organisms. In cells, sensing of increased levels of reactive oxygen species (ROS) activates many defensive mechanisms that limit or repair damage to cell components. The ROS-signaling responses necessary for cell survival under oxidative stress conditions remain incompletely understood, especially for the translational machinery. Here, we found that drug treatments or a genetic deficiency in the thioredoxin system that increase levels of endogenous hydrogen peroxide in the yeast Saccharomyces cerevisiae promote site-specific endonucleolytic cleavage in 25S ribosomal RNA (rRNA) adjacent to the c loop of …

Uncovering The Identity And Metabolism Of Bacterial Coa-Rna, Joseph R. Spangler

Dissertations

Coenzyme A is an indispensable molecule in all known life with roles in metabolism, gene regulation, and macromolecule synthesis. As CoA is derived from RNA itself, it’s incorporation into RNA by in vitro methods has proven useful in research probing the origin of life based on the RNA World theory. The discovery in contemporary bacteria of RNA modified with CoA, however, provided an unexpected twist to previously well-characterized bacterial systems. The identity of sequences associated with CoA-RNA has been elusive since their discovery in 2009 based on the difficulties in isolation while maintaining RNA quality. The aim of this study …

A Lipopolysaccharide-Induced Dna-Binding Protein For A Class Ii Gene In B Cells Is Distinct From Nf-Kappa B, Ellen M. Gravallese, Mark R. Boothby, Cynthia M. Smas, Laurie H. Glimcher

Ellen M. Gravallese

Class II (Ia) major histocompatibility complex molecules are cell surface proteins normally expressed by a limited subset of cells of the immune system. These molecules regulate the activation of T cells and are required for the presentation of antigens and the initiation of immune responses. The expression of Ia in B cells is determined by both the developmental stage of the B cell and by certain external stimuli. It has been demonstrated previously that treatment of B cells with lipopolysaccharide (LPS) results in increased surface expression of Ia protein. However, we have confirmed that LPS treatment results in a significant …

Regulation Of Synaptogenesis By The Mirna Pathway And Fmr/P Bodies, Jacqueline Rochelle Furlong

Electronic Theses and Dissertations

Post-transcriptional regulation of mRNA is facilitated by different mechanisms, such as microRNA (miRNA) induced gene silencing or fragile X mental retardation protein (FMRP) mediated repression either independent of or acting through cytoplasmic RNA Processing bodies (P bodies). DPTP99A, Lar, and Wg have known functions during synaptogenesis and may be targets of miR-8. Here, we provide evidence that miR-8 regulates DPTP99A in vitro. Non-endogenous miR-8 expressed using an UAS driver regulates Lar. Endogenous miR-8 may regulate DPTP99A in vivo. Here we show that FMRP is capable of colocalizing with the P body components: DCP1, HPat, and Me31B, but not …

Partitioning The Fitness Components Of Rna Populations Evolving In Vitro, Carolina Diaz Arenas, Niles Lehman

Chemistry Faculty Publications and Presentations

All individuals in an evolving population compete for resources, and their performance is measured by a fitness metric. The performance of the individuals is relative to their abilities and to the biotic surroundings – the conditions under which they are competing – and involves many components. Molecules evolving in a test tube can also face complex environments and dynamics, and their fitnessmeasurements should reflect the complexity of various contributing factors as well. Here, the fitnesses of a set of ligase ribozymes evolved by the continuous in vitroevolution system were measured. During these evolution cycles there are three different catalytic …

The Drosophila Melanogaster Rad54 Homolog, Dmrad54, Is Involved In The Repair Of Radiation Damage And Recombination, Rolf Kooistra, José B. M. Zonneveld, Anja De Jong, Jan C. J. Eeken, Chris J. Osgood, Jean-Marie Buerstedde, Paul H. M. Lohman, Albert Pastink

Biological Sciences Faculty Publications

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRAD54-deficient flies develop normally, but the females …

In Vitro Enzymatic Assay Of Rna Methylation, Pamela Jean Eubanks Gallup

Chemistry & Biochemistry Theses & Dissertations

An assay procedure for in vitro enzymatic methylation of mannnalian ribosomal RNA has been developed in this study. The assay procedure, utilized for the comparison of normal and neoplastic methylase activities (using mouse liver and Ehrlich ascites cells as sources of enzyme), is a modification of previously published methods (52,53). A 100,000 x g supernatant (SlOO) enzyme preparation was incubated with 28S-5.8S rRNA and tritium-labeled S-adenosyl-L-methionine. The RNA was extracted, applied to DEAE cellulose paper, washed, and the radioactivity counted. The neoplastic cell methylase preparation was more active in methylating both exogenous neoplastic and normal 28S-5.8S rRNA than the normal …