Browsing by Subject "Post-transcriptional regulation"
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Item Open Access Characterization of a Full-Length TTP Family Member Association with RNA Sequence Elements(2016) Washington, Onica LeighPost-transcriptional regulation of cytoplasmic mRNAs is an efficient mechanism of regulating the amounts of active protein within a eukaryotic cell. RNA sequence elements located in the untranslated regions of mRNAs can influence transcript degradation or translation through associations with RNA-binding proteins. Tristetraprolin (TTP) is the best known member of a family of CCCH zinc finger proteins that targets adenosine-uridine rich element (ARE) binding sites in the 3’ untranslated regions (UTRs) of mRNAs, promoting transcript deadenylation through the recruitment of deadenylases. More specifically, TTP has been shown to bind AREs located in the 3’-UTRs of transcripts with known roles in the inflammatory response. The mRNA-binding region of the protein is the highly conserved CCCH tandem zinc finger (TZF) domain. The synthetic TTP TZF domain has been shown to bind with high affinity to the 13-mer sequence of UUUUAUUUAUUUU. However, the binding affinities of full-length TTP family members to the same sequence and its variants are unknown. Furthermore, the distance needed between two overlapping or neighboring UUAUUUAUU 9-mers for tandem binding events of a full-length TTP family member to a target transcript has not been explored. To address these questions, we recombinantly expressed and purified the full-length C. albicans TTP family member Zfs1. Using full-length Zfs1, tagged at the N-terminus with maltose binding protein (MBP), we determined the binding affinities of the protein to the optimal TTP binding sequence, UUAUUUAUU. Fluorescence anisotropy experiments determined that the binding affinities of MBP-Zfs1 to non-canonical AREs were influenced by ionic buffer strength, suggesting that transcript selectivity may be affected by intracellular conditions. Furthermore, electrophoretic mobility shift assays (EMSAs) revealed that separation of two core AUUUA sequences by two uridines is sufficient for tandem binding of MBP-Zfs1. Finally, we found evidence for tandem binding of MBP-Zfs1 to a 27-base RNA oligonucleotide containing only a single ARE-binding site, and showed that this was concentration and RNA length dependent; this phenomenon had not been seen previously. These data suggest that the association of the TTP TZF domain and the TZF domains of other species, to ARE-binding sites is highly conserved. Domains outside of the TZF domain may mediate transcript selectivity in changing cellular conditions, and promote protein-RNA interactions not associated with the ARE-binding TZF domain.
In summary, the evidence presented here suggests that Zfs1-mediated decay of mRNA targets may require additional interactions, in addition to ARE-TZF domain associations, to promote transcript destabilization and degradation. These studies further our understanding of post-transcriptional steps in gene regulation.
Item Open Access MicroRNA Function in Cellular Stress Response(2012) Sangokoya, Carolyn OlufunmilayoMicroRNAs are key post-transcriptional regulators that have been found to play critical roles in the regulation of cellular functions. There is an emerging concept that microRNAs may be just as essential for fine-tuning physiological functions and responding to changing environments and stress conditions as for viability or development. In this dissertation, two studies are presented: The first study demonstrates a role for microRNA in the regulation of oxidative stress response in erythroid cells and the functional consequences of dysregulated microRNA expression in Sickle Cell Disease (SCD) pathobiology. The second study examines a functional role for microRNA in the cellular response to changes in cellular iron concentration. Together these studies illustrate the scope of importance of microRNAs in the coordination of cellular responses to diverse stresses.
Homozygous Sickle Cell (HbSS) erythrocytes are known to have reduced tolerance for oxidative stress, yet the basis for this phenotype has remained unknown. Here we use erythrocyte microRNA expression profiles to identify a subset of HbSS patients with higher miR-144 expression and more severe anemia. We reveal that in K562 erythroid cells and primary erythroid progenitor cells, miR-144 directly regulates NRF2, a central regulator of cellular response to oxidative stress, and modulates the oxidative stress response. We further demonstrate that increased miR-144 is associated with the reduced NRF2 levels, decreased glutathione regeneration, and attenuated antioxidant capacity found in HbSS erythroid progenitors, thereby providing a mechanism for the reduced oxidative stress tolerance and increased anemia severity seen in HbSS patients.
The post-transcriptional regulation of the IRP2 regulon in the cellular response to iron deficiency is well characterized. Here we examine the potential role for microRNA-mediated regulation in the coordinated response to cellular iron deficiency.
Item Open Access Post-transcriptional Regulation of Cancer Traits and Gene Expression in a Genetically Defined, Primary Cell-derived Model of Breast Tumorigenesis(2017) Bisogno, Laura SimonePost-transcriptional events are crucial determinants of gene expression, and aberrant expression patterns resulting from misregulation are evident in many pathological states. Cancer has traditionally been viewed as being driven by aberrant transcriptional regulation and signaling events, though, over the past several years, many RNA binding proteins and non-coding RNAs have emerged as critical players in tumor development. It is now recognized that regulation of post-transcriptional processes, such as mRNA stability and translation, robustly influence cancer-related gene expression patterns of proto-oncogenes, growth factors, cytokines, and cell cycle regulators. Despite its recognized importance, mechanisms of post-transcriptional regulation that influence molecular pathways at the mRNA level are understudied in the context of tumorigenesis. Additionally, cancer cells are derived from normal cells that often evolve step-wise and progressively to a neoplastic state, and the involvement of post-transcriptional regulation has not been looked at in the context of tumor initiation and step-wise progression. Thus, more studies are needed in order to fully understand the post-transcriptional mechanisms activated by cancer driver mutations that coordinate tumor initiation and progression.
In this dissertation, we aimed to elucidate mechanisms of post-transcriptional regulation coordinating tumorigenesis. We first established a genetically defined, primary cell-derived model of breast cancer initiation and progression. In this model, normal human mammary epithelial cells were immortalized through the expression of hTERT, p53DD, cyclin D1, CDK4R24C and c-MYCT58A, and subsequently converted to a tumorigenic state through expression of oncogenic H-RASG12V. Using RNA-sequencing and real-time PCR arrays, we comprehensively quantified changes in mRNA abundance, miRNA expression and alternative splicing in this system, and revealed thousands of changes during immortalization and relatively few changes during RAS transformation. Moreover, pre-malignant, immortalized cells had expression signatures consistent with an epithelial-to-mesenchymal transition (EMT), but they expressed low levels of mesenchymal protein markers and were non-invasive. Activation of RAS in these pre-malignant cells induced an invasive phenotype without major changes in global mRNA expression. Consistent with post-transcriptional mechanisms, RAS increased protein levels of Vimentin and N-cadherin without changing mRNA levels.
We then sought to investigate a mechanism of this RAS-induced post-transcriptional regulation. We used a method developed in our lab called Digestion-Optimized Ribonucleoprotein Immunoprecipitation coupled with RNA-sequencing (DO-RIP-seq) to identify and quantify transcriptome-wide binding sites for the RNA binding protein HuR. Our study is the first to identify and quantify transcriptome wide binding sites for any RBP during tumorigenesis, and we report that HuR quantitatively, but not qualitatively, changed association at individual mRNA binding sites during RAS transformation. We identified a GU-rich secondary motif associated with a decrease in HuR binding during transformation. Furthermore, our data suggest that HuR may cooperate with the CELF1 protein to positively regulate the translation of a subset of mRNAs and promote the EMT phenotype. We generated HuR CRISPR knockout cell lines and demonstrated that HuR expression was necessary for the maintenance of cancer traits, including proliferation, anchorage independent growth, migration and invasion, but it does not regulate mRNA stability in this context. Lastly, we identified a binding site position dependent mechanism by which HuR regulates alternative polyadenylation of mRNAs encoding proteins involved in cancer-related processes.
In conclusion, our findings indicate that EMT-associated invasion can be initiated through two sequential stages: transcriptional priming followed by oncogenic RAS-triggered post-transcriptional regulation. The HuR RNA binding protein is important for maintaining the cancer phenotypes induced by oncogenic RAS, and regulation by HuR may be, at least in part, determined by a GU-rich secondary motif as well as cooperation with the CELF1 RNA binding protein.
Item Open Access Post-transcriptional regulation of gene expression in response to iron deficiency in Saccharomyces cerevisiae(2010) Vergara, Sandra VivianaThe ability of iron (Fe) to easily transition between two valence states makes it a preferred co-factor for innumerable biochemical reactions, ranging from cellular energy production, to oxygen transport, to DNA synthesis and chromatin modification. While Fe is highly abundant on the crust of the earth, its insolubility at neutral pH limits its bioavailability. As a consequence, organisms have evolved sophisticated mechanisms of adaptation to conditions of scarce Fe availability.
Studies in the baker's yeast Saccharomyces cerevisiae have shed light into the cellular mechanisms by which cells respond to limited Fe-availability. In response to Fe-deficiency, the transcription factors Aft1 and Aft2 activate a group of genes collectively known as the Fe-regulon. Genes in this group encode proteins involved in the high-affinity plasma membrane Fe-transport and siderophore uptake systems, as well as Fe-mobilization from intracellular stores and heme re-utilization. Concomitant with the up-regulation of the Fe-regulon, a large number of mRNAs encoding Fe-dependent proteins as well as proteins involved in many Fe-dependent processes are markedly down regulated. Thus, in response to low Fe-levels the cell activates the Fe-uptake and mobilization systems, while down-regulating mRNAs involved in highly Fe-demanding processes leading to a genome-wide remodeling of cellular metabolism that permits the funneling of the limiting Fe to essential Fe-dependent reactions.
The Fe-regulon member Cth2 belongs to a family of mRNA-binding proteins characterized by an RNA-binding motif consisting of two tandem zinc-fingers of the CX8CX5CX3H type. Members of this family recognize and bind specific AU-rich elements (AREs) located in the 3'untranslated region (3'UTRs) of select groups of mRNAs, thereby promoting their rapid degradation. In response to Fe-limitation, Cth2 binds ARE sequences within the 3'UTRs of many mRNAs encoding proteins involved in Fe-homeostasis and Fe-dependent processes, thereby accelerating their rate of decay.
Work described in this dissertation demonstrates that the Cth2 homolog, Cth1, is a bona fide member of the Fe-regulon, binds ARE-sequences within the 3'UTRs of select mRNAs and promotes their decay. Cth1 and Cth2 appear to be only partially redundant; Cth1 preferentially targets mRNAs encoding mitochondrial proteins, while Cth2 promotes the degradation of most of Cth1 targets in addition to other mitochondrial and non-mitochondrial Fe-requiring processes. The coordinated activity of Cth1 and Cth2 results in dramatic changes in glucose metabolism. In addition, experiments described in this dissertation indicate that the CTH1 and CTH2 transcripts are themselves subject to ARE-mediated regulation by the Cth1 and Cth2 proteins, creating an auto- and trans-regulatory circuit responsible for differences in their expression. Finally, work described here demonstrates that Cth2 is a nucleocytoplasmic shuttling protein and that shuttling is important for the early determination of cytosolic mRNA-fate.
Item Open Access Posttranscriptional Regulation of Embryonic Neurogenesis by the Exon Junction Complex(2016) Mao, HanqianThe six-layered neuron structure in the cerebral cortex is the foundation for human mental abilities. In the developing cerebral cortex, neural stem cells undergo proliferation and differentiate into intermediate progenitors and neurons, a process known as embryonic neurogenesis. Disrupted embryonic neurogenesis is the root cause of a wide range of neurodevelopmental disorders, including microcephaly and intellectual disabilities. Multiple layers of regulatory networks have been identified and extensively studied over the past decades to understand this complex but extremely crucial process of brain development. In recent years, post-transcriptional RNA regulation through RNA binding proteins has emerged as a critical regulatory nexus in embryonic neurogenesis. The exon junction complex (EJC) is a highly conserved RNA binding complex composed of four core proteins, Magoh, Rbm8a, Eif4a3, and Casc3. The EJC plays a major role in regulating RNA splicing, nuclear export, subcellular localization, translation, and nonsense mediated RNA decay. Human genetic studies have associated individual EJC components with various developmental disorders. We showed previously that haploinsufficiency of Magoh causes microcephaly and disrupted neural stem cell differentiation in mouse. However, it is unclear if other EJC core components are also required for embryonic neurogenesis. More importantly, the molecular mechanism through which the EJC regulates embryonic neurogenesis remains largely unknown. Here, we demonstrated with genetically modified mouse models that both Rbm8a and Eif4a3 are required for proper embryonic neurogenesis and the formation of a normal brain. Using transcriptome and proteomic analysis, we showed that the EJC posttranscriptionally regulates genes involved in the p53 pathway, splicing and translation regulation, as well as ribosomal biogenesis. This is the first in vivo evidence suggesting that the etiology of EJC associated neurodevelopmental diseases can be ribosomopathies. We also showed that, different from other EJC core components, depletion of Casc3 only led to mild neurogenesis defects in the mouse model. However, our data suggested that Casc3 is required for embryo viability, development progression, and is potentially a regulator of cardiac development. Together, data presented in this thesis suggests that the EJC is crucial for embryonic neurogenesis and that the EJC and its peripheral factors may regulate development in a tissue-specific manner.
Item Open Access Structural Characterization of the Bacterial Riboregulator Hfq and the Novel M. tuberculosis Toxin-Antitoxin Module Rv3188-Rv3189(2017) Kovach, Alexander RobertThe bacterial protein Hfq is an RNA chaperone and pleiotropic posttranscriptional regulator. Hfq binds to A and U-‐‑rich regions of small regulatory RNA (sRNA) to their cognate mRNA to facilitate their annealing, affecting stability and translation. The protein is involved in the regulation of a wide array of cellular processes, including many related to environmental stress response and virulence. The importance of Hfq in Gram-‐‑negative bacteria is well understood, while a less clear picture remains for Gram-‐‑positive species. We have determined the structure of Hfq from the Gram-‐‑positive pathogen Listeria monocytogenes (Lm) in its apo form and bound to U6 RNA. U6 RNA binds to the proximal face in a canonical manner but with additional contacts made to the N3 and O4 positions of uridine by residue Q6 of Hfq. Furthermore, fluorescence polarization and tryptophan fluorescence quenching (TFQ) reveal that U16 RNA binds to Hfq with higher affinity than U6, on the basis of the longer sequence’s ability to simultaneously bind in the proximal pore and the lateral rim of the protein. TFQ also shows that surprisingly Lm Hfq can accommodate (GU)3G and U6 RNA on both proximal and distal face binding sites, suggesting Lm Hfq has a less stringent distal face A-‐‑site than previously reported for Hfq from other species.
To understand fully how sRNA bind to the proximal face and are positioned to anneal with mRNA, we have attempted to crystallize U16 RNA with Lm Hfq and fragments of sRNA containing a hairpin with a poly-‐‑U tail with both Lm and Escherichia coli (Ec) Hfq. While this endeavor has been largely fruitless, we have determined the structure of Ec Hfq with dsDNA. Ec Hfq-‐‑DNA binding has been observed in multiple studies but the molecular mechanism of recognition of this nucleic by Hfq is unknown. The DNA binds to the proximal face with conserved lateral rim residues N13, R16, and R17, and residue Q41 contacting the phosphate backbone. Fluorescence polarization and TFQ reveal both dsDNA and dsRNA bind to the proximal face, indicating the observed DNA binding mode may actually be a double stranded nucleic acid binding site. We have thusly proposed a model in which the proximal face of Hfq stabilizes both single and double stranded portions of sRNA, positioning it appropriately for formation of an Hfq-‐‑sRNA-‐‑mRNA ternary complex.
Toxin-‐‑antitoxin (TA) modules are ubiquitous among bacterial species with bioinformatics studies identifying at least 10000 putative TA modules. These modules have diverse functions and are implicated in many processes, including gene regulation, stress response, and persister cell formation. Whereas many bacteria may have only a handful of TA modules, the genome of Mycobacterium tuberculosis (Mtb) contains 79 TA modules, 37 of which have been confirmed to be functional in vivo. A recent transcriptome analysis of Mtb persister cells revealed 10 up-‐‑regulated TA modules. Four of these modules do not belong to a previously characterized TA family. We have determined the structure of a C-‐‑terminally truncated version of the toxin Rv3189 (1-‐‑164 of 206 amino acid residues) in complex with the anti-‐‑toxin Rv3188. Rv3189 is structurally homologous to the ADP-‐‑ribosyltransferase core domain, suggesting a never before observed mode of action for a TA module. The toxin has been shown to inhibit growth in an E. coli model with structure guided mutagenesis identifying residues that are critical for toxin function.