Both genetic and environment factors influence susceptibility to substance use disorders. However, the genetic basis of these disorders is largely unknown. We previously identified Hnrnph1 (heterogeneous nuclear ribonucleoprotein H1) as a quantitative trait gene for reduced methamphetamine (MA) stimulant sensitivity. Mutation (heterozygous deletion of a small region in the first coding exon) in Hnrnph1 also decreased MA reinforcement, reward, and dopamine release. 5’UTR genetic variants in Hnrnph1 support reduced 5’UTR usage and hnRNP H protein expression as a molecular mechanism underlying the reduced MA-induced psychostimulant response. Interestingly, Hnrnph1 mutant mice show a two-fold increase in hnRNP H protein in the striatal synaptosome with no change in whole tissue level. Proteome profiling of the synaptosome identified an increase in mitochondrial complex I and V proteins that rapidly decreased with MA in Hnrnph1 mutants. In contrast, the much lower level of basal mitochondrial proteins in the wild-type mice showed a rapid, MA-induced increase. Altered mitochondrial proteins associated with the Hnrnph1 mutation may contribute to reductions in MA behaviors. hnRNP H1 is an abundant RNA-binding protein in the brain, involved in all aspect of post-transcriptional regulation. We examined both baseline and MA-induced changes in hnRNP H-RNA interactions to identify targets of hnRNP H that could comprise the neurobiological mechanisms of cellular adaptations occurring following MA exposure. hnRNP H post-transcriptionally regulates a set of mRNA transcripts in the striatum involved in psychostimulant-induced synaptic plasticity. MA treatment induced opposite changes in binding of hnRNP H to these mRNA transcripts between Hnrnph1 mutants versus wild-types. RNA-binding, transcriptome, and spliceome analyses triangulated on hnRNP H binding to the 3’UTR of Cacna2d2, an upregulation of Cacna2d2 transcript, and decreased 3’UTR usage of Cacna2d2 in response to MA in the Hnrnph1 mutants. Cacna2d2 codes for a presynaptic, voltage-gated calcium channel subunit that could plausibly regulate MA-induced dopamine release and behavior. The multi-omics datasets point to a dysregulation of mitochondrial function and interrelated calcium signaling as potential mechanisms underlying MA-induced dopamine release and behavior in Hnrnph1 mutants.

No description available.

Post-transcriptional regulations (PTRs) have always been considered features of organisms with higher complexity. However recently, the interest toward the post- transcriptional mechanisms in prokaryotes increased. The bacterial proteome is much more complex compared to the genome size, suggesting a tight and articulate regulation of proteins production, extremely important for the bacterial adaptation to an always changing environment. Bacterial PTRs are responsible of modulation of mRNA stability and decay, translation initiation and elongation, modulation of the access of ribosome to the ribosome binding site and control of termination of the transcript. The main actors in the PTRs are small non-coding RNA (responsible of the inhibition of the transcription) and RNA binding proteins (RBPs), which modulate the translation and half-life of the mRNA. RBPs, are particularly of my interest since I wanted to find a possible orthologous of the eukaryotic Elav-like (Elavl) family of proteins in Acinetobacter baumannii. Elav-like proteins are present in all metazoans and are characterized by two highly conserved sequences: RNP-1 (a quite well conserved hexamer) and RNP-2 (a really well conserved octamer) that are responsible of binding to the mRNA. Each species has a different number of Elavl paralogous that is totally independent from the complexity of the organisms, suggesting a more ancient origin. In particular, I focused on the human paralog HuR (human antigen R). HuR is characterized by three RNA Recognition motif (RRM) -domains, is ubiquitously expressed and is mainly localized into the nucleus (where it is responsible of maturation of the mRNA), but under stress stimuli, can shuttle into the cytoplasm where protect the target mRNA from degradation, by binding AU/U rich sequences (ARE sequences). Its high concentration into the cytoplasm can lead to the overexpression of oncogenes and pro-tumorigenic factors. The choice of Acinetobacter baumannii comes from the increasing worldwide concern toward this pathogen that is becoming multidrug resistant. Indeed, in Italy, more the 50% of nosocomial infections are caused by A. baumannii. I found a putative protein (AB-Elavl), composed by a single RRM domain endowed with similar features of the eukaryotic RRM domain as the presence of a quite well conserved RNP-2 and a less conserved RNP-1. I expressed this protein with recombinant tools and confirmed the production of the protein in the host by western blot and mass spectrometry. I evaluated the binding activity of AB-Elavl testing the EC50 and the Kd with different biochemical assays (EMSA, AlphaScreen and HTRF- FRET) toward three different RNA sequences, in order to test the specificity. By X- RAY and NMR, I confirmed the folded structure that can be overlapped to the HuR’s one and the interaction with the probes tested, highlighting the presence of binding, but with different specificity. I also tested some small molecules developed for interfering in the binding of HuR with the target sequence and found a possible compound able to interact with AB-Elavl, by disrupting the binding with the target probe. All these results suggest an ancient origin of the metazoans’ Elavl family of proteins that probably share a common ancestor with AB-Elavl. More studies should be performed to better understand the role of AB-Elavl in A. baumannii as well as in other bacteria. In fact, I found the presence of other ARE sequence-binding proteins also in Pseudomonas aeruginosa. Interesting would be to check the presence of this protein in all the multidrug resistant ESKAPE bacteria.

No description available.

No description available.

[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. / [CA] 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]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/55941

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

The proper expression of a gene to a protein is a complicated process with many steps. One of the major steps is translation, the process of decoding a messenger RNA signal and the building of a protein from its component parts. The control of translation is one of the major steps for the overall control of gene expression and its dysregulation is associated with a wide variety of human diseases including neurological, metabolic and reproductive disorders. Dazl family proteins are germ cell restricted RNA binding proteins that contain a motif characteristic of this family, the DAZ domain. Whilst humans encode all three family members DAZ, DAZL and BOULE, flies only possess the boule gene. The members of this family have an essential conserved role in gametogenesis in a wide variety of organisms from worm to man with loss of function resulting in phenotypes ranging from male or female infertility or both. However, little is known about the molecular role of these proteins in germ cell development. A previous study within the laboratory showed that several vertebrate Dazl family members can stimulate translation of a reporter gene in Xenopus laevis oocytes, suggesting a conserved role in mRNA specific translational control. This is consistent with studies in invertebrates. It was proposed that Dazl proteins fulfil this function through an interaction with a translation initiation factor, poly(A) binding protein, PABP. The aim of this thesis was to further refine this model of action. The work presented here investigates several fundamental questions regarding the mechanism of Dazl-mediated stimulation. First, it investigated the step of translation initiation that Dazl acts upon and explored the initiation factors that may be required. Second, it addressed in more detail the requirements for an interaction between Dazl and the poly(A) binding protein, PABP. Third, it examined the potential role of another factor, DAZ associated protein 1, DAZAP1, in Dazl-mediated stimulation. The role of multi-protein complexes containing Dazl bound to the 3’UTR that localise, repress and stimulate translation of specific mRNAs at defined times during gametogenesis are discussed.

Positive strand RNA viruses are intracellular parasites, and their genome replication and infection involves complex virus-host interactions. Therefore, identification of host factors and dissection of their functions during virus replication could facilitate our understanding of the mechanism of virus infection. Those host factors may also provide new targets for viral disease control. Tomato bushy stunt virus (TBSV) has recently become one of the model viruses to study positive strand RNA virus replication and hostvirus interactions. To identify host factors involved in TBSV replication we used yeast as a model host. Co-expression of the replication proteins and a replicon RNA (DI RNA) via plasmids in yeast resulted in robust replication of the viral RNA. Previous work using a yeast single gene deletion library (YKO) revealed 96 yeast genes affecting virus replication. The essential yeast genes could not be deleted so we used the Yeast Tet Promoters Hughes Collection (yTHc) where the original promoter was replaced by Tetracyclin-titratable promoter. I tested the 800 essential host genes available in yTHc. In total, we found 30 new host genes whose down-regulated expression either increased or decreased the accumulation of a TBSV repRNA. The identified essential yeast genes fall into different categories on the basis of the cellular processes they are involved in, such as RNA transcription/metabolism, protein metabolism/transport etc. Detailed analysis of the effects of some of the identified yeast genes revealed that they might affect RNA replication by altering (i) the amounts of p33 and p92(pol) viral replication proteins, (ii) the activity of the tombusvirus replicase complex, and (iii) the ratio of plus- versus minus-stranded RNA replication products. Altogether, this and previous YKO screening of yeast led to the identification of 126 host genes (out of ~5,600 genes that represent ~95% of all the known and predicted yeast genes) that affected the accumulation of tombusvirus RNA. In the YKO screening, we found NSR1 (homologous to plant nucleolin) gene, whose deletion led to increased TBSV repRNA accumulation. Nucleolin is an abundant RNA binding protein, which shuttles between the nucleolus, the nucleoplasm and the cytoplasm. This protein is involved in rRNA maturation, ribosome assembly and regulation of cellular RNA metabolism.We found that over-expression of Nsr1p in yeast or nucleolin in Nicotiana benthamiana inhibited the accumulation of tombusvirus RNA by ~10-fold. Temporal regulation of Nsr1p over-expression revealed that the inhibitory effect of Nsr1p was more profound when it was expressed at early stages of viral replication. In vitro binding experiments showed that Nsr1p binds preferably to the RIII in the repRNA (which is derived from 3’ UTR of viral genome). Consistent with its RIII specific binding, over-expression of Nsr1p only reduced 40% of the accumulation of TBSVΔRIII repRNA in yeast. The purified recombinant Nsr1p inhibited the in vitro replication of the viral RNA in a yeast cell-free assay when pre-incubated with the viral RNA before the in vitro replication assay. Our data suggest that Nsr1p/nucleolin inhibits tombusvirus replication by interfering with the recruitment of the viral RNA for replication.

<p>It is well-established that the organization of nuclear components influences gene expression processes, yet little is known about the mechanisms that contribute to the spatial co-ordination of nuclear activities. The salivary gland cells of <i>Chironomus tentans</i> provide a suitable model system for studying gene expression<i> in situ</i>, as they allow for direct visualization of the synthesis, processing and export of a specific protein-coding transcript, the Balbiani ring (BR) pre-mRNA, in a nuclear environment in which chromatin and non-chromatin structures can easily be distinguished. The RNAbinding protein Hrp65 has been identified in this model system as a protein associated with non-chromatin nucleoplasmic fibers, referred to as connecting fibers (CFs). The CFs associate with BR RNP particles in the nucleoplasm, suggesting that Hrp65 is involved in mRNA biogenesis at the post-transcriptional level. However, the function of Hrp65 is not known, nor is the function or the composition of CFs. In the work described in this thesis, we have identified by yeast two-hybrid screening and characterized different proteins that bind to Hrp65. These proteins include a novel hnRNP protein in <i>C. tentans</i> named Hrp59, various isoforms of Hrp65, the splicing- and mRNA export factor HEL/UAP56, and a RING-domain protein of unknown function. Immuno-electron microscopy experiments showed that Hrp59 and HEL are present in CFs, and in larger structures in the nucleoplasm of <i>C. tentans</i> salivary gland cells.</p><p>Hrp59 is a <i>C. tentans</i> homologue of human hnRNP M, and it associates cotranscriptionally with a subset of pre-mRNAs, including its own transcript, in a manner that does not depend quantitatively on the amount of synthesized RNA. Hrp59 accompanies the BR pre-mRNA from the gene to the nuclear envelope, and is released from the BR mRNA at the nuclear pore complex. We have identified the preferred RNA targets of Hrp59 in <i>Drosophila</i> cells, and we have shown that Hrp59 binds preferentially to exonic splicing enhancer sequences.</p><p>Hrp65 self-associates through an evolutionarily conserved domain that can also mediate heterodimerization of Hrp65 homologues. Different isoforms of Hrp65 interact with each other in all possible combinations, and Hrp65 can oligomerize into complexes of at least six molecules. The interaction between different Hrp65 isoforms is crucial for their intracellular localization, and we have discovered a mechanism by which Hrp65-2 is imported into the nucleus through binding to Hrp65-1.</p><p>Hrp65 binds to HEL/UAP56 in <i>C. tentans</i> cells. We have analyzed the distribution of the two proteins on polytene chromosomes and in the nucleoplasm of salivary gland cells, and our results suggest that Hrp65 and HEL become associated during posttranscriptional gene expression events. HEL binds to the BR pre-mRNP cotranscriptionally, and incorporation of HEL into the pre-mRNP does not depend on the location of introns along the BR pre-mRNA. HEL accompanies the BR mRNP to the nuclear pore and is released from the BR mRNP during translocation into the cytoplasm.</p>