Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 109-133).

No description available.

With the number of fully sequenced bacterial genomes increasing in the past 7 years, it has been discovered that a large percentage of the putative protein coding genes have no known function. This lack of knowledge leaves scientists with an incomplete understanding of bacteria. In this study, conserved hypothetical protein mutants from Streptococcus sanguinis SK36 were screened on solid media with various environmental conditions. From these screens, the candidate protein, SSA_2372, displayed a sensitivity to acidic conditions. Its homolog in Bacillus subtilis 168, BSU00030, also displayed a sensitivity to pH conditions at its acid tolerance extremes unlike its other homolog in Escherichia coli, YbcJ. When the growth rate and cell yield was acquired, the sensitivity was shown to be significant for both SSA_2372 and BSU00030 mutants. Through data mining, it was determined that Firmicutes in this homolog family COG2501 may function as a regulator for recombination protein F.

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.

In skeletal muscle acetylcholinesterase AChE is highly expressed at sites of nerve-muscle contact where it is regulated at both the transcriptional and post-transcriptional levels. Scientists have elucidated many aspects of synaptic AChE structure, function, and localization during the past 80 years. However our understanding of the molecular mechanisms underlying its regulation is incomplete, but it appears to involve both translational and post-translational events as well. We found that Pumilio-2 (PUM2), an RNA binding translational repressor, is highly localized at the neuromuscular junction where AChE mRNA concentrates and that PUM2 binds to the AChE transcripts when immoprecipitation studies were performed. A direct binding between a recombinant PUM2-HD and the Pumilio Binding Site (PBE) in a segment of the AChE 3’UTR was demonstrated by Gel shift assays. Transfecting skeletal muscle cells with shRNAs specific for PUM2 upregulated AChE expression, whereas overexpression of PUM2 decreased AChE activity. We conclude that PUM2 binds to AChE mRNA and regulates AChE expression translationally at the neuromuscular synapse. We found that PUM2 is regulated by the motor nerve suggesting a trans-synaptic mechanism for locally regulating translation of specific synaptic proteins involved in modulating synaptic transmission, analogous to CNS synapses. PUM2 expression is critically important in many cell types, virtually nothing is known about the regulation of PUM2 expression itself. Analyzing the PUM2 mRNA 3’UTR we found fifteen possible PBEs in the 3 Kb 3’ UTR. We show that PUM2 binds in vivo to its own mRNA. Overexpression of PUM2 in several cell types transfected with a green fluorescent protein (GFP) reporter construct linked to the full length PUM2 3’UTR (GFP-PUM2-3’UTRFL) suppresses GFP expression suggesting that PUM2 downregulates its own expression by binding to its own 3’UTR. Mutations of the first five PBEs yield the expression of the reporter gene indicating that at least one PBE is functional in the autoregulation of PUM2. These observations suggest a novel model for the localized regulation of protein translation through a negative feedback loop. Much is known about PUM2 as a translational regulative protein but little is known about PUM2 cell localization and possible mechanism of translational regulation. In this work we found PUM2 to be highly localized to the cell rough endoplasmic reticulum and that PUM2 is associated with ribosomal RNA. In addition, we found that the GFP protein itself, together with its mRNA and ribosomal RNA (rRNA), were localized in the PUM2 positive complexes when GFP-PUM2-3’UTRFL was transfected into muscle cells. These observations further suggest a mechanism of regulation where translation of the protein occurs but the protein remains associated with the ribonucleoprotein complex, possibly to be transported together with its mRNA to specific domains inside the cell. Thus when needed, more protein is produced in those specific cell regions.

Ribosomes which conduct protein synthesis in all living organisms are comprised of two subunits. The large 60S ribosomal subunit catalyzes peptidyl transferase reactions and includes the polypeptide exit tunnel, while the small (40S) ribosomal subunit recruits incoming messenger RNAs (mRNAs) and performs proofreading. The plant 80S cytoplasmic ribosome is composed of 4 ribosomal RNAs (rRNAs: 25-28S, 5.8S and 5S in the large subunit and 18S in the small subunit) and 81 ribosomal proteins (r-proteins: 48 in the large subunit, 33 in the small subunit). RPS15a, a putative small subunit primary binder, is encoded by a six member gene family (RPS15aA-F), where RPS15aB and RPS15aE are evolutionarily distinct and thought to be incorporated into mitochondrial ribosomes. In vitro synthesized cytoplasmic 18S rRNA, 18S rRNA loop fragments, and RPS15a mRNA molecules were combined in electrophoretic shift assays (EMSAs) to determine the RNA binding characteristics of RPS15aA/-D/-E/-F. RPS15aA/F, -D and -E bind to cytoplasmic 18S rRNA in the absence of cellular components. However, RPS15aE r-protein tested that binds mitochondrial 18S rRNA. In addition, RPS15aA/F only binds one of three 18S rRNA loop fragments of helix 23 whereas RPS15aD/-E bind all three 18S rRNA helix 23 loop fragments. Additionally, RPS15aD and RPS15aE did not bind their respective mRNA transcripts, likely indicating that this form of negative feedback is not a post-transcriptional control mechanism for this r-protein gene family. Furthermore, the addition of RPS15a transcripts to the EMSAs did not affect the binding of RPS15aA/F, -D and -E to 18S rRNA helix 23 loop 4-6, indicating that rRNA binding is specific. Supershift EMSAs further confirmed the specificity of RPS15aA/F and RPS15aE binding to loop fragment (4-6) of 18S rRNA. Taken together, these data support a role for RPS15a in early ribosome small subunit assembly.

Ribosomes which conduct protein synthesis in all living organisms are comprised of two subunits. The large 60S ribosomal subunit catalyzes peptidyl transferase reactions and includes the polypeptide exit tunnel, while the small (40S) ribosomal subunit recruits incoming messenger RNAs (mRNAs) and performs proofreading. The plant 80S cytoplasmic ribosome is composed of 4 ribosomal RNAs (rRNAs: 25-28S, 5.8S and 5S in the large subunit and 18S in the small subunit) and 81 ribosomal proteins (r-proteins: 48 in the large subunit, 33 in the small subunit). RPS15a, a putative small subunit primary binder, is encoded by a six member gene family (RPS15aA-F), where RPS15aB and RPS15aE are evolutionarily distinct and thought to be incorporated into mitochondrial ribosomes. In vitro synthesized cytoplasmic 18S rRNA, 18S rRNA loop fragments, and RPS15a mRNA molecules were combined in electrophoretic shift assays (EMSAs) to determine the RNA binding characteristics of RPS15aA/-D/-E/-F. RPS15aA/F, -D and -E bind to cytoplasmic 18S rRNA in the absence of cellular components. However, RPS15aE r-protein tested that binds mitochondrial 18S rRNA. In addition, RPS15aA/F only binds one of three 18S rRNA loop fragments of helix 23 whereas RPS15aD/-E bind all three 18S rRNA helix 23 loop fragments. Additionally, RPS15aD and RPS15aE did not bind their respective mRNA transcripts, likely indicating that this form of negative feedback is not a post-transcriptional control mechanism for this r-protein gene family. Furthermore, the addition of RPS15a transcripts to the EMSAs did not affect the binding of RPS15aA/F, -D and -E to 18S rRNA helix 23 loop 4-6, indicating that rRNA binding is specific. Supershift EMSAs further confirmed the specificity of RPS15aA/F and RPS15aE binding to loop fragment (4-6) of 18S rRNA. Taken together, these data support a role for RPS15a in early ribosome small subunit assembly.

<p>Much of the regulation of gene expression occurs at the posttranscriptional level, and much of this regulation is controlled and coordinated by RNA binding proteins (RBPs). Many RBPs have multiple mRNA targets, and the proteins encoded by these targets often share functional relationships, forming posttranscriptional RNA operons. These operons often reflect the function of the RBP, thus determination of the genome-wide targets of RBPs allows insight into their functions.</p> <p>The PUF family of RBPs is characterized by the presence of an extremely well conserved RNA binding domain, typically consisting of 8 repeats of an RNA binding motif, with each repeat binding to one RNA base. PUF proteins are proposed to have an ancestral role in self-renewal of stem cells and have been shown to affect a number of developmental processes. Human and other vertebrate genomes contain two canonical PUF genes, Pum1 and Pum2, and at the outset of this study there was very little known about functions or targets of either protein, especially Pum1.</p> <p>In order to identify the genome-wide targets of human Pum1 we used RNA immunoprecipitation followed by microarray, or RIP-Chip, analysis. RIP-Chip allowed us to identify Pum1 target mRNAs in human HeLa cells. We found that there were numerous functional relationships among the proteins encoded by these mRNAs, forming putative RNA operons. Some of these potential operons are progression of cell cycle, cell differentiation and proliferation, and regulation of transcription. We were also able to find a consensus Pum1 binding motif, UGUAHAUA, in the 3' UTRs of Pum1 target mRNAs. </p> <p>The genome-wide targets of PUF proteins from other species have been previously identified, and by comparing the targets of human Pum1 to targets of Drosophila Pumilio and yeast Puf3, both of which bind to the same RNA sequence as Pum1, we determined that there has been evolutionary rewiring of regulation by Puf proteins. While the PUF RNA binding domain and consensus binding sequence have remained almost identical through evolution, the surrounding protein sequence and the mRNAs bound have changed dramatically, indicating that evolutionary rewiring is occurring in a modular fashion. </p> <p>After identifying Pum1 associated mRNAs, we went on the study the function of Pum1. Through Pum1 knockdown assays we found that Pum1 enhances decay of target mRNAs, and that this effect is likely due to Pum1 enhancing deadenylation of these mRNAs. We also showed by immunofluorescence that Pum1 protein has a cytoplasmic granular subcellular localization and upon oxidative stress relocates to stress granules but not processing bodies. We were, however, unable to detect any difference in Pum1 mRNA targeting after stress. We were also unable to detect any changes in progression through cell cycle after Pum1 knockdown. </p> <p>In this study we identified the genome-wide mRNAs associated with Pum1, determined functional relationships among these targets related to the proposed ancestral role of PUF proteins in self-renewal of stem cells, and identified a sequence motif to which Pum1 binds in these mRNAs. We also demonstrated that Pum1 enhances decay of associated mRNAs, and that this effect is likely due to Pum1 enhancing deadenylation of associated mRNAs. These results provide a description of mRNA targets and mechanisms of action of Pum1 proteins, which will provide a strong foundation for future experiments to further explore the functions of the Pum1, especially as they relate to human stem cells.</p> / Dissertation

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 127-139).

Cardiac muscle function necessitates the meticulous assembly and interactions of several cytoskeletal and regulatory proteins into specialized structures that orchestrate contraction and transmission forces. Despite extensive studies identifying the protein components responsible for these important aspects of heart development, putative RNA based mechanisms remain poorly understood, even with their demonstrated importance in other tissues. Evidence suggests that post-transcriptional regulation is critical for muscle function, but the molecular players involved (RNA binding proteins and mRNA targets) have remained elusive. We investigated the molecular mechanisms and targets of the muscle-specific Fragile X Related protein-1 (FXR1), an RNA binding protein whose absence leads to perinatal lethality in mice. Loss of FXR1 results in global protein level alterations. Morphological and biochemical analyses of Fxr1^(-/-) mice revealed severe disruption of intercalated disc and costamere architecture and composition. We identified several candidate mRNAs specifically enriched in the FXR1 protein complex. Two targets that likely contribute to the architectural defects are desmoplakin (dsp) and talin2 (tln2). In vitro assays indicate that FXR1 binds to these mRNA targets directly and represses their translation. Additionally, we provide preliminary evidence that the Fxr1^(-/-) mice mimic a hypothyroid state of cardiac gene expression, with alterations in myosin heavy chain and troponin I isoforms. Our findings reveal the first mRNA targets of FXR1 in muscle and support translational repression as a novel mechanism for cardiac muscle development and function.