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Thesis advisor: Welkin Johnson / Thesis advisor: Michelle Meyer / Cis-regulatory RNA elements are structured regions of an mRNA that regulate the transcription, translational efficiency, or stability of the mRNA. These cis-regulatory RNAs are widely used across all domains of life to modulate gene expression in response to various stimuli. In bacteria, examples of these cis-regulatory RNAs include small RNAs, structured 50-500 nucleotide non-coding RNA that bind to mRNA or protein to alter expression, and riboswitches, which consist of a ligand-binding aptamer domain whose complex tertiary structure selectively responds to specific ligands to regulate downstream gene expression on the transcriptional or translational level. Ribosomal protein expression in bacteria is often controlled using an autogenous cis-regulatory mechanism, in which select ribosomal proteins (r-proteins) bind RNA structures in the 5’-untranslated of their own mRNA to regulate the expression of r-protein operons. Some of these structures, such as the RNA leaders regulating r-proteins L1, L20, and S2, have striking homology and often mimicry between the recognition motifs within their primary binding partner, ribosomal RNA (rRNA), and their secondary binding partner, the structured mRNA leader. Ribosomal protein S15 is a notable exception to this trend, as the five regulatory RNA leaders identified across various bacterial species that respond to S15 are structurally distinct, narrowly distributed to their respective phyla, and often bear little obvious homology to the rRNA. Additionally, inter-species interaction studies have shown that the S15 homologs from these species have specific recognition profiles fori the mRNA regulators, and not all interactions are reciprocal. How RNA regulators arise and are maintained in bacterial genomes is not well understood, and thus we sought to use ribosomal protein S15 as a model to study how differences in the RNA-binding profiles of the various S15 homologs may have driven the diversity of the mRNA regulators we see today. To explore these RNA-binding profiles, I utilized an in vitro selection approach to enrich for aptamers (structured RNAs that bind a specific ligand) that bind the S15 homologs from Escherichia coli (EcS15), Geobacillus kaustophilus (GkS15), and Thermus thermophilus (TtS15) from a partially patterned RNA sequence pool. Following multiple attempts to enrich for aptamers to EcS15, I find that aptamers to this homolog are infrequent in this RNA sequence pool. I successfully enriched for Gk- and TtS15 aptamers from this sequence pool and using high-throughput sequencing and clustering analysis go on to show that these homologs have highly overlapping RNA-binding profiles, though the aptamers enriched by TtS15 exhibit slightly more sequence diversity than those enriched by GkS15. I confirm that three unique aptamers from the final RNA pools bind both homologs in vitro, and a single nucleotide change that differentiates two of these aptamers causes a decrease in affinity for TtS15 but not GkS15. This mutation causes a change in the predicted folding of these two aptamers, and greatly reduces its frequency in the population enriched by TtS15. Taken together, the work presented in this thesis shows overlapping but not identical RNA-binding profiles for the Gk- and TtS15 homologs to aptamers enriched from a partially patterned RNA library and represents the first comparative study of two homologous RNA-binding proteins using in vitro selection against an RNA library. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

The ability of a cell to respond precisely to environmental stress depends on the expression of a large number of genes in a finely coordinated manner. One of such genes is CHOP that encodes the CCAAT/Enhancer-Binding Protein Homologous Protein. CHOP is usually expressed to mediate apoptosis under the condition of excessive stress. The expression of CHOP therefore has to be stringently regulated as its expression will determine the fate of a cell under stress. The expression of many genes is regulated at the posttranscriptional level through the metabolism of their mRNA, such as maturation, transport, storage, and degradation of mRNA. Many metabolic processes of mRNA are known to be mediated by RNA-binding proteins that specifically interact with the mRNA. RNA-binding proteins that interact with the CHOP mRNA have until present not been identified. The aim of this study is to investigate what proteins may bind specifically to CHOP mRNA. The study will enable further understanding regarding how the expression of CHOP is regulated in cellular stress response. Proteins extracted from HeLa cells were incubated with a 335bp [3H]-labelled CHOP RNA probe that spans over a part of the coding region and the 3’UTR of CHOP mRNA. Sucrose density gradient ultracentrifugation revealed that after incubation with proteins extracted from HeLa cells, the sedimentation rate of the [3H]-CHOP RNA probe was significantly higher than that of the free [3H]-RNA probe. The formation of heavy molecular complexes involving the [3H]-CHOP RNA probe was therefore suggested. However, no increase in sedimentation rate of the [3H]-CHOP RNA probe was observed in the presence of an excess of unlabelled CHOP RNA probe. Similar observations were made when the experiments were performed using proteins isolated from cells treated with As2O3. Two putative sequence elements, the Adenylate-Uridylate-Rich Element (ARE) and the Putative Regulatory Element (PRE) located respectively in the 3’UTR and coding region of the CHOP mRNA were then examined for their involvement in RNA-protein interaction. The deletion of ARE and/or PRE, from the [3H]-CHOP RNA probe had little effect on the binding of the RNA probe to the HeLa cell proteins. Consistently, unlabelled CHOP RNA probes with the same deletions were only slightly weaker in competing with the intact [3H]-CHOP RNA probe to bind to HeLa cell proteins. Human Antigen R (HuR) was identified by Western blot analysis to be present in the proteins that were obtained by pull-down assays using biotinylated CHOP RNA as a probe. The deletion of ARE and/or PRE resulted in a slight reduction of HuR obtained by pull down assays. This study provides the first evidence that physical binding interaction occurs between intracellular RNA-binding proteins and CHOP mRNA. More importantly, one such protein is HuR. Data suggest that HuR binding to the CHOP mRNA is mediated by sequences in the CHOP mRNA other than ARE and PRE. / published_or_final_version / Biochemistry / Master / Master of Philosophy

In addressing the pre-mRNA substrate, the splicing machinery requires rearrangement of multiple RNA and protein components. The classical model of spliceosome formation begins with the U1 snRNA recognition of the 5" splice site and U2 snRNP interaction with the branch point. This process is followed by the engagement of a pre-assembled U4/U6·U5 tri-snRNP to form the A2-1 complex. The spliceosome is subsequently activated through a number of structural rearrangements. Among these is the unwinding of the U4/U6 intermolecular helix by the tri-snRNP component Brr2p. While numerous protein components of the tri-snRNP have been identified, the function of many of these remain unknown. The nonessential Snu66p (U4/U6·U5-110K in humans) stably associates only with the U4/U6·U5 tri-snRNP while the similarly nonessential Snu40p (U5-52K in humans) associates exclusively with the U5 snRNP. To understand why two non-essential pre-mRNA splicing factors have been so well conserved through great evolutionary distances, we examined their roles in the assembly and function of the tri-snRNP. Removal of SNU40 alone does not affect snRNP levels, however deletion of SNU66 results in reduced levels of tri-snRNP. The U4/U6·U5 snRNPs in [Delta]snu66 cells are resistant to the ATP-dependent U4/U6 unwinding by Brr2p, and profound U4/U6 accumulation occurs at reduced temperatures. Remarkably, subsequent removal of SNU40 in a [Delta]snu66 strain bypasses the tri-snRNP formation defect while unwinding of U4/U6 remains defective. Additional investigation revealed that Prp6p, another tri-snRNP protein, is destabilized from the complex. Based upon this data in total, I present a model in which Snu40p and Snu66p interact sequentially with Prp6p to maintain directionality for proper biogenesis of the tri-snRNP. Further, the U4/U6 unwinding defect of the double mutant should theoretically arrest the A2-1 spliceosome. Indeed, native gel analysis confirms the buildup of a large complex later determined to be A2-1. I have purified this complex, functionally tested its catalytic viability, and identified its components via mass spectrometry. This is the first full characterization of the A2-1 precatalytic spliceosome complex in Saccharomyces cerevisiae. / text

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