The RNA-binding protein HuR is involved in numerous aspects of the RNA life-cycle. It is known for its ability to stabilize AU-Rich Element (ARE)-containing transcripts in the cytoplasm. The transcript of cIAP1, an important protein involved both in the regulation of apoptosis and NF-κB signaling, contains four such AREs, raising the question of whether HuR can modulate the stability of cIAP1 mRNA. First, using C2C12 cells, we observed a positive correlation between cIAP1 mRNA levels and HuR cytoplasmic localization. We then show that knockdown of HuR in U2OS cells results in a decrease in steady-state cIAP1 mRNA levels through destabilization of the cIAP1 mRNA. Furthermore, we are able to show in vitro that HuR binds directly to the second of the four AREs in the 3’ UTR. The direct link between the binding of HuR to the second ARE and its effect on cIAP1 mRNA stability remains to be shown.

<p>The 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. </p> <p>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. </p> <p>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. </p> <p>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.</p> / Dissertation

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24833号 / 医博第5001号 / 新制||医||1067(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田, 宏一, 教授 上野, 英樹, 教授 濵﨑, 洋子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

G-quadruplexes, characterized by stacked G-tetrad rings held together by Hoogsteen hydrogen bonds, have been visualized in human cells and implicated in transcriptional and translational control, telomere maintenance and disease. RHA Helicase associated with AU-rich element (RHAU), a DEAH-box helicase, is a major G-quadruplex resolvase in human cell lysates. It binds G-quadruplexes through the RHAU specific motif in its N-terminus. In order to investigate the recognition of G-quadruplexes by helicases, the binding between the N-terminal construct of RHAU, RHAU53-105, and the DNA analog of the quadruplex formed by the 5’ terminus of human telomerase RNA component, hTR1-20, was investigated in a comprehensive biophysical approach followed by crystallization screening. RHAU53-105, hTR1-20 DNA and their complexes were analysed by gel electrophoresis, UV-visible spectroscopy, spectropolarimetry, dynamic light scattering and small angle X-ray scattering (SAXS). The findings reveal that hTR1-20 DNA, separated in two conformations by size exclusion chromatography in the presence of potassium cations, assumes a disk-like parallel G-quadruplex secondary structure in solution. Far-UV circular dichroism spectra and SAXS demonstrate that RHAU53-105 assumes an extended (Dmax = 7.8 nm , rG = 2.1 (±0.2) nm) and ordered conformation in solution. The analysis confirms the binding between RHAU53-105 and each conformation of the hTR1-20 DNA quadruplex. Circular dichroism spectra indicate the retention of quadruplex secondary structure in both RHAU53-105•hTR1-20 DNAc1 and RHAU53-105•hTR1-20 DNAc2 complexes. This analysis provides some insight into the interaction between G-quadruplexes and the N-terminal domain of RHAU and identifies 0.2 M sodium formate, 20 % (w/v) polyethylene glycol 3350 and 1.5 M sodium chloride, 10 % (v/v) ethanol as preliminary conditions for crystallization of the complex of RHAU53-105 and hTR1-20 DNAc2. / October 2014

No description available.

Regulation of gene expression occurs at several levels in a cell. While control of transcription is often viewed as the main source of regulation, it is now clear that post-transcriptional processes are essential to fine-tune protein availability. The presence of AU-rich elements (ARE) in the 3’ untranslated region (3’UTR) of many important mRNAs exemplifies one such process. AREs alter the mRNA translation or degradation status by recruiting ARE-binding proteins (ARE-BP). ARE-BPs of the TTP/TIS11 family bind to their cognate ARE-RNAs using their conserved tandem zinc-finger domain and induce rapid decay of their targets. This allows for proper regulation of cell proliferation, cell death and inflammation. In this regard, TTP/TIS11 are main regulators of gene expression, and as such are put under strict transcriptional, post-transcriptional as well as several layers of post-translational control.<p>In this work, we aimed at elucidating the degradation mechanisms affecting TTP/TIS11. Using Drosophila as a model, we found that dTIS11 protein turnover is rapid due to continuous degradation by the proteasome. However, proteasomal recognition did not require ubiquitination of dTIS11 as non-ubiquitinable mutants were efficiently degraded by the proteasome. In addition, dTIS11 was digested by the 20S proteasome that lacks ubiquitin-recognition domains. Our results further indicate that intrinsically disordered regions (IDR) in dTIS11 may be responsible for proteasomal recognition. In fact, dTIS11 is predicted as disordered and possesses the main characteristics of intrinsically disordered proteins (IDP). We also identified dTIS11 N- and C-terminal domains as functional signals for degradation, potentially due to their destructuration. This ubiquitination-independent, disorder-dependent degradation process is conserved throughout evolution as dTIS11 mammalian counterpart, TTP, undergoes the same degradation by default pathway. In addition, we established that phosphorylation prevents degradation of TTP/TIS11 by the proteasome. <p>Together, our results pinpoint a new essential characteristic for TTP/TIS11 that may redefine the identity of these proteins. In addition, we unraveled a novel and conserved mechanism of regulation of TTP/TIS11. This control is essential for cell physiology as defects in this process can lead to defects in the inflammatory response, increased radiation-induced lung toxicity and tumorigenesis.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished