The RNA-binding protein HuR controls key cellular processes by binding target mRNAs and regulating them at various post-transcriptional levels. HuR can function as an Internal Ribosome Entry Site (IRES) trans-acting factor that regulates the IRES-mediated translation of XIAP. Since XIAP and Bcl-xL expression was reported to be co-regulated, we investigated whether HuR is also a regulat or of Bcl-xL expression. We found that HuR binds the 3’end of the Bcl-xL 5’UTR in-vitro. In U2OS cells, we showed that loss of HuR by siRNA significantly increased Bcl-xL protein expression while Bcl-2 and Mcl-1 levels remained unchanged. We found that the HuR-dependent Bcl-xL increase was through translation, shown by polysome profiling. Possible transcriptional, stability and splicing changes were eliminated. At the physiological level HuR levels did not impact cell survival but altered mitochondrial morphology, partially through Bcl-xL. Thus, HuR may be involved in maintaining proper mitochondrial function by controlling Bcl-xL expression.

Differentiation is the process of regulated gene expression that gives rise to different phenotypes from a common genotype. Skeletal muscle differentiation, myogenesis, is a good example of this process. Skeletal muscle is susceptible to injury due to direct or indirect causes. If left unrepaired, these injuries may lead to a loss of muscle mass, locomotive deficiency, and even lethality. Thus, understanding the molecular mechanisms behind this process is an important first step in the design of treatment for muscle-related diseases. Once myogenesis is induced, the expression of MRF proteins, such as MyoD and myogenin, is maintained at high levels in myofibers without the need to increase their rates of transcription, suggesting a role for post-transcriptional regulatory mechanisms. HuR is a ubiquitously expressed member of the embryonic lethal, abnormal vision (ELAV) family of RNA binding proteins that is known to post-transcriptionally regulate its target messages. Here, I demonstrate that, in the C2C12 muscle cell line, HuR is a required factor for both the initiation and maintenance of the myogenic process. First in vitro RNA Electro-Mobility Shift Assays (REMSA) and immunoprecipitation experiments demonstrated that HuR specifically binds to the AU-rich elements (AREs) that are present in the 3' untranslated regions (3'UTRs) of the MyoD and myogenin mRNAs. In the absence of HuR at the time of differentiation induction, accomplished using the siRNA technology, the expression of the MyoD and myogenin messages is significantly reduced, leading to inhibition of myogenesis. At this early stage in the differentiation process, HuR, a shuttling protein, is predominantly nuclear; localization that is mediated by the import receptor Transportin2 (Trn2). Nuclear HuR was determined to be required for the negative regulation of nucleophosmin (NPM) translation. Forced overexpression of NPM, resulting in differentiation inhibition, shows that its downregulation is a requirement for induction of the differentiation process. Late in myogenesis, however, NPM RNA is no longer expressed, and HuR is seen to accumulate in the cytoplasm of myotubes. This cytoplasmic accumulation results from dissociation of HuR from Trn2 due to caspase-dependent cleavage within its HNS region. Specifically blocking HuR import through the use of cell-permeable peptides, as well as RNAi-mediated depletion of Trn2, leads to an increase in cytoplasmic HuR, as well as increased cytoplasmic localization and stabilization of the MyoD and myogenin messages, and a corresponding enhancement of differentiation. Overall, we conclude that HuR is required for myogenesis due to its ability to post-transcriptionally regulate genes required for the process, and that HuR itself is regulated at the level of its subcellular localization, mediated by the import receptor TRN2.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Thesis (M.S.)--University of Missouri--Columbia, 2003. / "December 2003." Typescript. Includes bibliographical references (leaves 37-38). Also issued on the Internet.

Autophagy is compromised in Parkinson’s disease (PD) with a number of PD-associated genetic mutations leading to its dysregulation. Leucine-rich repeat kinase (LRRK2) mutations, causative of PD, aberrantly enhance autophagy. Our lab elucidated a LRRK2 gene regulatory network identifying transcripts showing coordinated expression level changes associated with PD. Histone deacetylase 6 (HDAC6) was found to be an important interactor with LRRK2, regulating many of the same transcripts. The majority of these transcripts associate with autophagy and the lysosomal complex. I hypothesized that LRRK2 interacts with HDAC6 to regulate autophagy. Silencing of HDAC6 in SH-SY5Y normalized the autophagosomal size altered by expression of PD-linked LRRK2 mutants. This work identified a key role for HDAC6 in mediating the autophagic dysfunction induced by the mutant LRRK2. In addition to autophagy, stress granule (SG) formation has emerged as a compelling mechanism in the pathogenesis of PD. RNA-binding proteins (RBPs), such as T-cell intracellular antigen-1 (TIA-1), are major component of SGs. I observed TIA-1 translocating from the nucleus to the cytoplasm in PD cortex without forming SGs. Hu antigen D (HuD) also showed changes, with the RBP more present in the cytoplasm than the nucleus in PD with no SGs observed. These preliminary studies lead to the hypothesis that low levels of SGs result from an inhibition by alpha-synuclein (syn), or hyperactive autophagy. For that purpose, brain tissues from a mouse model of PD (A53T-syn transgenic mouse) were examined by immunohistochemistry. There was no difference in TIA-1 expression in control and A53T-syn expressing mouse brains, or SG formation in primary neurons after treatment with recombinant A53T fibrils. To determine whether the lack of SGs in PD brain was due to activation of autophagy, BE-M17 cells were treated with rapamycin, an autophagy activator, which decreased SGs by 50%. Overexpression of TIA-1 in BE-M17 cells under arsenite treatment also increased autophagosomal size by 50%, indicating co-regulation of SGs and autophagy. My work indicates that the pathophysiology of PD is associated with a loss of SGs due to elevated activity of autophagy, presumably due to PD-linked LRRK2 mutations. This co-regulatory network may be a potential therapeutic target of PD.

Abnormal aggregation of microtubule associated protein tau is the defining pathological hallmark of tauopathies, which include Alzheimer’s disease (AD) and related frontotemporal dementias (FTLD-tau). However, the cellular events precipitating tau pathogenesis in disease are unknown. Here, we demonstrate a novel mechanism regulating tau aggregation in tauopathies. We have previously shown that RNA binding proteins (RBPs) associated with stress granules (SGs) progressively accumulate with tau in multiple mouse models of tauopathy, as well as in human AD and FTLD-tau brain tissue. We now present a novel functional role for tau in regulating the biology of SGs in neurons. Tau facilitates the rapid formation of SGs in the soma and dendrites in response to exogenous stress, which functions to transiently reprogram protein synthesis to promote cell survival (also known as the ‘translational stress response’). However, the chronic interaction of tau with SG proteins in disease, such as with the SG nucleating protein T cell intracellular antigen 1 (TIA1), promotes tau misfolding and neurotoxicity, which can be modulated in primary neurons by pharmacological or genetic manipulations that increase (i.e. puromycin, TIA1 overexpression) or decrease (i.e. cycloheximide, TIA1 knockdown or knockout) SG formation, respectively. In order to test whether SGs also mediate the progression of tauopathy in vivo, we crossed PS19 transgenic (P301S) tau mice with Tia1-/- or C57BL/6J (background strain) mice. PS19 mice with heterozygous reduction in TIA1 (P301S TIA1+/-) developed less SGs compared to P301S TIA1+/+ mice, which was associated with marked neuronal protection, improved cognitive function, and prolonged lifespan. The behavioral neuroprotection in P301S TIA1+/- mice was associated with decreased accumulation of soluble tau oligomers, and occurred despite the increased presence of neurofibrillary tangles. Our findings suggest that TIA1 stabilizes tau in its oligomeric state, preventing its further assembly into insoluble fibrils, which are less toxic. More importantly, the studies described in this dissertation identify modulation of RBP aggregation in SGs as a promising therapeutic strategy for the treatment of AD and FTLD-tau.

xv, 106 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / Myotonic Dystrophy type I (DM1) is caused by muscleblind protein sequestration to aberrantly expanded CUG repeats. When muscleblind is sequestered it can no longer fulfill its role as an alternative splicing regulator, leading to mis-splicing events in both humans and Drosophila . The muscleblind protein family's RNA binding specificity has been minimally characterized. Only one pre-mRNA target in humans, cardiac troponin T (cTNT), has a known MBNL1 binding site. In order to understand muscleblind's RNA binding specificity and identify a consensus binding motif, systematic evolution of ligands by exponential enrichment (SELEX) was performed on both the Drosophila muscleblind protein, Mbl, and the human ortholog, MBNL1. Drosophila has provided a useful model for studying the disease mechanism of DM1. Studies of Mbl's RNA binding specificity to CUG repeats concluded that replacing the U-U mismatches with different pyrimidine-pyrimidine mismatches was tolerated, but no other mutations were. To understand Mbl's RNA binding specificity, SELEX was performed. After 6 rounds, several sequences were identified that bound with high affinity, all containing the 5'-AGUCU-3' consensus motif. One sequence, SELEX RNA 20 was analyzed further. In addition to the guanosine in the consensus motif of SELEX RNA 20, two other guanosines were shown to be protected by Mbl in a footprinting assay, indicating that Mbl has a strong preference for binding guanosine. Also, two "tail" regions of SELEX RNA 20 were shown to be single stranded and required for binding by Mbl. These results indicate that Mbl is a highly specific RNA binding protein with preference for both single and double stranded guanosine-rich regions. A doped SELEX was performed on MBNL1's binding site from the cTNT pre-mRNA to determine which sequences and structural aspects were important for recognition by MBNL1. Pool 5 RNA sequences bound with high affinity, and the motif 5'-YGCUU-3' was selected. This motif was then used to identify new MBNL1 binding sites in pre-mRNAs regulated by MBNL1, SERCA1 and MBNL1. The identification of this motif and two new MBNL1 sites provide insight into MBNL1-mediated alternative splicing. This dissertation includes both my previously published co-authored material and my unpublished co-authored material. / Adviser: J. Andrew Berglund

The function of two subunits of the putative mitochondrial RNA binding complex (MRB1) associated with RNA editing in parasitic protist Trypanosoma brucei was studied using various in vivo and in vitro methods of molecular biology.

The RNA-binding protein HuR controls key cellular processes by binding target mRNAs and regulating them at various post-transcriptional levels. HuR can function as an Internal Ribosome Entry Site (IRES) trans-acting factor that regulates the IRES-mediated translation of XIAP. Since XIAP and Bcl-xL expression was reported to be co-regulated, we investigated whether HuR is also a regulat or of Bcl-xL expression. We found that HuR binds the 3’end of the Bcl-xL 5’UTR in-vitro. In U2OS cells, we showed that loss of HuR by siRNA significantly increased Bcl-xL protein expression while Bcl-2 and Mcl-1 levels remained unchanged. We found that the HuR-dependent Bcl-xL increase was through translation, shown by polysome profiling. Possible transcriptional, stability and splicing changes were eliminated. At the physiological level HuR levels did not impact cell survival but altered mitochondrial morphology, partially through Bcl-xL. Thus, HuR may be involved in maintaining proper mitochondrial function by controlling Bcl-xL expression.

[EN] Ribosome biogenesis is one of the most important and energy-consuming processes in the cell. However, the vast majority of the events and factors that are involved in the synthesis of ribosomal subunits are not well understood. Ribosome maturation comprises multiple steps of rRNA processing that require sequential association and dissociation of numerous assembly factors. These proteins establish a complex network of interactions that are essential for the pathway to continue. Extensive studies in Saccharomyces cerevisiae allowed to identify some of the genetic and functional correlations between the pre-ribosomal factors that could be organized into interdependent clusters or sub-complexes. A heterotrimer formed by Nop7, Erb1 and Ytm1 (PeBoW complex in mammals) is crucial for the proper formation of the 60S subunit. Depletion of any of the three proteins is inviable and certain truncations result in aberrant processing of 27SA2 rRNA thus impairing cell proliferation. Nop7 and Erb1 have been shown to bind RNA and are recruited to the pre60S before Ytm1. It is also known that the trimer has to be removed from the nascent particle in order to promote its normal maturation. Despite its relevance in the cell, the exact role of PeBoW is not clear and the interactions within the complex have been poorly characterized. In this study we carry out an extensive biochemical and structural analysis of Nop7-Erb1-Ytm1 trimer from S. cerevisiae and from a thermophilic fungus Chaetomium thermophilum. We have been able to reconstitute a stable complex in vitro that was then used in crystallographic trials. We have solved the structure of the C-terminal domain of Erb1 from yeast that folds into a seven-bladed ß-propeller. We prove that this part of the protein binds RNA in vitro, a property that might be important for its function. Moreover, in spite of previous reports suggesting that the ß-propeller domain of Erb1 would not be essential for ribosome biogenesis, we could solve the crystal structure of Ytm1 bound to the carboxy-terminal portion of Erb1 from C. thermophilum. That finding led us to redefine the macromolecular interactions that hold the complex together. First, we have verified that the N-terminal region of Nop7 interacts with Erb1. Furthermore, we have shown that a good affinity binding takes place in vitro between WD40 domain of Ytm1 and the ß-propeller of Erb1. Upon careful analysis of the interface involved in dimer formation we have designed a mutant of Erb1 that exhibits weaker association with Ytm1. We confirm our structural and biophysical data using S. cerevisiae. We prove that a point mutation that decreases the affinity between propellers of Erb1 and Ytm1 negatively affects growth in yeast because it interferes with 60S production. We show that a very conserved interface of protein-protein interaction could be targeted in order to hinder cell proliferation. / [ES] El ensamblaje de ribosomas es uno de los procesos más importantes y costosos energéticamente en una célula eucariota. A pesar de ello, se sabe relativamente poco acerca de la gran mayoría de los eventos y factores implicados en la síntesis de las subunidades ribosomales. La maduración de ribosomas comprende numerosos pasos de procesamiento del rRNA que requieren la asociación y disociación de más de doscientos factores de ensamblaje. Esas proteínas establecen una compleja red de interacciones que son esenciales para que el proceso pueda llevarse a cabo. Los estudios realizados en Saccharomyces cerevisiae han permitido la identificación de algunas correlaciones genéticas y funcionales entre los factores prerribosomales. Es el caso del heterotrímero formado por Nop7, Erb1 e Ytm1 (complejo PeBoW en mamíferos), que es imprescindible para la correcta formación de la subunidad 60S. La ausencia de cualquiera de las tres proteínas es inviable y también se conocen ciertas variantes truncadas que alteran el procesamiento del rRNA 27SA2 y de este modo afectan la proliferación celular. Se ha demostrado que Nop7 y Erb1 se asocian al rRNA y que su reclutamiento al pre60S ocurre antes de la unión a Ytm1. Además se sabe que el trímero tiene que separarse de la partícula prerribosomal emergente con el fin de favorecer su maduración. A pesar de su gran relevancia en la célula, no está claro el papel exacto del complejo PeBoW y tampoco se dispone de conocimientos suficientes acerca de las interacciones intermoleculares que lo mantienen. Durante el desarrollo de este proyecto se ha llevado a cabo un exhaustivo análisis bioquímico y estructural del trímero Nop7-Erb1-Ytm1 procedente de S. cerevisiae y del hongo termofílico Chaetomium thermophilum. En este trabajo hemos sido capaces de reconstituir el complejo estable in vitro que posteriormente se ha utilizado en los ensayos de cristalización, con los que hemos podido resolver la estructura del dominio carboxi-terminal de Erb1 de levadura, cuyo plegamiento corresponde a una hélice enrollada (ß-propeller) de siete hojas. Gracias a la información estructural, hemos demostrado que esa parte de la proteína es capaz de unir RNA in vitro, lo que puede ser una propiedad importante para su función. Además, a pesar de los estudios anteriores que sugerían que la hélice enrollada de Erb1 no era esencial en la biogénesis del ribosoma, hemos resuelto la estructura cristalina de la proteína Ytm1 unida al dominio C-terminal de Erb1 de C. thermophilum. Ese descubrimiento nos ha permitido redefinir las interacciones macromoleculares que mantienen el complejo. Inicialmente hemos confirmado que el extremo amino-terminal de Nop7 interacciona con Erb1. A continuación, hemos demostrado que el dominio WD40 de Ytm1 se une al ß-propeller de Erb1 con una buena afinidad. Después de un detallado análisis de la superficie involucrada en la formación del dímero, hemos sido capaces de diseñar una variante mutada de Erb1 que se asocia más débilmente con Ytm1. Los hallazgos estructurales y biofísicos se han confirmado in vivo usando S. cerevisiae donde hemos demostrado que una mutación puntual que disminuye la afinidad de unión entre los dominios C-terminales de Erb1 e Ytm1 manifiesta un efecto negativo sobre el crecimiento de levadura porque interfiere con la síntesis de 60S. Nuestros resultados establecen un buen ejemplo de una superficie conservada involucrada en interacciones proteína-proteína, que podría considerarse una buena diana para inhibir la proliferación celular eucariota. / [CAT] L'ensamblatge de ribosomes és un dels processos més importants i energèticament costosos en una cèl·lula eucariota. Tot i això, es coneix relativament poc de la majoria dels factors implicats en la síntesi de les subunitats ribosomals. La maduració de ribosomes compren moltes etapes de processament del rRNA que requereix l'associació i dissociació de més de dos-cents factors d'ensamblatge. Aquestes proteïnes estableixen una complexa xarxa de interaccions que són essencials perquè el procés es pugi dur a terme. Els estudis realitzats en Saccharomyces cerevisiae han permès la identificació de algunes correlacions genètiques i funcionals entre els factors pre-ribosomals. Aquest és el cas del heterotrímer comprés per Nop7, Erb1 i Ytm1 (complex PeBoW en mamífers), que és imprescindible per a la correcta formació de la subunitat 60S. L'absència de qualsevol de les tres proteïnes és inviable i també és coneixen certes variants truncades que alteren el processament del rRNA 27SA3 i que d'aquesta manera afecten a la proliferació cel·lular. S'ha demostrat que Nop7 i Erb1 s'associen al rRNA i que el seu reclutament al pre60S té lloc abans de l'unió a Ytm1. A més a més, es sap que el trímer ha de separar-se de la partícula pre-ribosomal emergent per tal que es produeixi la seua maduració. Malgrat la seua rellevància en la cèl·lula, no s'ha aclarit el paper exacte del complex PeBoW i tampoc n'hi ha coneixements suficients de les interaccions intermoleculars que el mantenen. Durant el desenvolupament d'aquest projecte s'ha dut a terme un exhaustiu anàlisi bioquímic i estructural del trímer Nop7-Erb1-Ytm1 de S. cerevisiae i del fong termofílic Chaetomium thermophilum. En aquest treball hem estat capaços de reconstituir el complex estable in vitro que posteriorment s'ha utilitzat en el assajos de cristal·lització, amb els que hem pogut resoldre l'estructura del domini carboxi-terminal de Erb1 de llevat i que té un plegament corresponent a una hèlix enrotllada (ß-propeller) de set fulles. Gràcies a la informació estructural, hem pogut demostrar que aquesta part de la proteïna té la capacitat d'unir RNA in vitro, el que pot ser una propietat important per a la seua funció. A més a més, malgrat que els estudis anteriors suggerien que la hèlix enrotllada de Erb1 no era essencial en la biogènesis del ribosoma, hem pogut resoldre la estructura cristal·lina de la proteïna Ytm1 unida al domini C-terminal de Erb1 de C. thermophilum. Aquest descobriment ens ha permès redefinir les interaccions macromoleculars que mantenen el complex. Inicialment, hem confirmat que l'extrem amino-terminal de Nop7 interacciona amb Erb1. A continuació, hem demostrat que el domini WD40 de Ytm1 s'uneix al ß-propeller de Erb1 amb bona afinitat. Després d'un anàlisi detallat de la superfície involucrada en la formació del dímer, hem estat capaços de dissenyar una variant mutada de Erb1 que s'associa més dèbilment amb Ytm1. Les dades estructurals i biofísiques s'han confirmat in vivo utilitzant S. cerevisiae on hem demostrat que una mutació puntual que disminueix l'afinitat d'unió entre els dominis C-terminals de Erb1 i Ytm1 manifesta un efecte negatiu en el creixement del llevat perquè interfereix amb la síntesi del 60S. Els nostres resultats estableixen un bon exemple de una superfície conservada involucrada en interaccions proteïna-proteïna, que es podria considerar una bona diana per a inhibir la proliferació cel·lular eucariota. / Wegrecki, M. (2015). Structural, biophysical and functional characterization of Nop7-Erb1-Ytm1 complex and its implications in eukaryotic ribosome biogenesis [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/55941 / TESIS