The heat-shock response is one of the many complex physiological systems that organisms have developed in order to protect their cells against stress. This response is initiated by the binding of heat shock factor 1 (HSF1) to the promoters of genes containing heat-shock elements (HSEs,) which results in the expression of several proteins, among them the proteo-protective inducible heat-shock protein (hsp70i). Due to HSF1s critical role in this process, an active area of research is trying to understand of how HSF1 executes its function. Considering the rapidity with which the field of cell biology is expanding, in particular the sub-field of nuclear compartmentalization, this study seeks to understand how nuclear structure affects the function of HSF1. Specifically, this study investigates the potential role for the interaction between HSF1 and the translocated promoter region protein (Tpr,) a structural component of the nuclear pore, an interaction initially identified by yeast two-hybrid analysis, in the transcription of hsp70i. Due to Tprs location and its putative function in nucleo-cytoplasmic trafficking, this works seeks to answer to the question, Does Tpr play a role in the export of HSF1-driven mRNAs? In a similar vein, heat-shock transcription factor 2 (HSF2,) a less well-understood member of the heat-shock transcription factor family, also interacts with Tpr in the yeast two-hybrid assay. HSF2 has recently been shown to have an active role during mitosis, when the hsp70i gene is being bookmarked for potential expression that might be needed in early G1, when most genes are unable to be expressed. This body of work also seeks to answer the question of, Does the Tpr/HSF2 interaction have a role in positioning the gene in relation to the nuclear pore after mitosis? This study was performed using both novel and standard in vivo and in vitro molecular biology techniques. It ultimately aims to clarify the less understood, although much broader, subject of how does transcription occur in the three-dimensional space of the nucleus.

No description available.

No description available.

<p>The unique C-terminal domain (CTD) of RNA polymerase II (RNAPII), composed of tandem heptad repeats of the consensus sequence YSPTSPS, is subject to differential phosphorylation throughout the transcription cycle. Several RNA processing factors have been shown to bind the appropriately phosphorylated CTD, and this facilitates their localization to nascent pre-mRNA during transcription. In <italic>Saccharomyces cerevisiae</italic>, the mRNA export protein Yra1 (ALY/REF in metazoa) has been shown to cotranscriptionally associate with mRNA and is thought to deliver it to the nuclear pore complex for export to the cytoplasm. Based on a previous proteomics screen, I hypothesized that Yra1 is a <italic>bona fide</italic> phosphoCTD associated protein (PCAP) and that this interaction is responsible for the pattern of Yra1 cotranscriptional association observed <italic>in vivo</italic>. Using <italic>in vitro</italic> binding assays, I show that Yra1 directly binds the hyperphosphorylated form of the CTD characteristic of elongating RNAPII. Using truncations of Yra1, I determined that its phosphoCTD-interacting domain (PCID) resides in the segment comprising amino acids 18-184, which, interestingly, also contains the RNA Recognition Motif (RRM) (residues 77-184). Using UV crosslinking, I found that the RRM alone can bind RNA, although a larger protein segment, extending to the C-terminus (aa 77-226), displays stronger RNA binding activity. Even though the RRM is implicated in both RNA and CTD binding, certain RRM point mutations separate these two functions: thus, mutations that produce defects in RNA binding do not affect CTD binding. Both functions are important <italic>in vivo</italic>, in that RNA binding-defective or CTD binding-defective versions of Yra1 engender growth and mRNA export defects. I also report the construction and characterization of a useful new temperature sensitive <italic>YRA1</italic> allele (<italic>R107AF126A</italic>). Finally, using chromatin immunoprecipitation, I demonstrate that removing the N-terminal 76 amino acids of Yra1 (all of the PCID up to the RRM) results in a 10-fold decrease in Yra1 recruitment to genes during elongation. These results indicate that the PCTD is likely involved directly in cotranscriptional recruitment of Yra1 to active genes.</p> / Dissertation

Influenza A virus (IAV) replication and transcription occur in the host cell nucleus; a feature which means both the viral genome (vRNA) and mRNA must be exported from the nucleus to the cytoplasm. The mechanism by which vRNA nuclear export is achieved has been well characterised, but how viral mRNAs are exported is poorly understood. The cellular NXF1-dependent mRNA export pathway has been shown to be involved in the export of some viral mRNAs, but how they are recruited to this pathway is unknown. Prior work from our laboratory showed that segment 7 mRNA was inefficiently exported to the cytoplasm in a sub-viral ‘minireplicon’ system, providing the first indication that there were viral requirements for IAV mRNA nuclear export. Further addition of individual viral polypeptides was tested and the effect on segment 7 mRNA export was analysed by fluorescent in situ hybridization (FISH) and confocal microscopy. This identified the NS1 protein as the viral factor required for efficient segment 7 nuclear export. Mutational studies on NS1 were carried out to unveil the mechanistic role of this protein in viral mRNA nuclear export, by plasmid transfection as well as in the context of recombinant viruses. These approaches indicated that both functional domains of NS1 were necessary to preserve the mRNA export function. Furthermore, these mutant proteins were used to examine the association between NS1 and the NXF1-dependent pathway in the context of mRNA nuclear export. Protein-protein and protein-RNA binding assays indicated that interactions between NXF1 and NS1, and NXF1 and segment 7 mRNA were necessary, but not sufficient to promote segment 7 viral mRNA export. Lastly, the role of NS1 protein in the nuclear export of viral mRNAs from other genome segments was studied. The intracellular localisation of most viral mRNAs was not affected by the absence of NS1 or the presence of an export-incompetent NS1 mutant protein. However, segment 4 mRNA exhibited a similar phenotype to segment 7 mRNA in showing a dependence on NS1 for efficient nuclear export. Overall, the results presented in this dissertation suggest that NS1 acts as an adaptor protein between the viral RNA synthesis machinery and cellular export pathway. This provides deeper insights for the characterization of a recently identified function of the IAV NS1 protein, of being required for the efficient nuclear export of mRNA from “late” kinetic class viral genes.

Nuclear export of messenger ribonucleic acid (mRNA) is a complex and essential process for a correct gene expression in all eukaryotic cells. The export of mRNA through the nuclear pore complex depends mostly on the crosstalk and coordination of several proteins forming what is known as mRNPs (messenger ribonucleoproteins) that play dynamic, interconnecting roles in the different mRNA biogenesis steps such as pre-mRNA processing, stability, and export. One key protein in this process is Mex67, conserved from yeast to humans, is the major messenger RNA exporter also involved in ribosomal RNA export. Mex67 interacts with Mtr2 to form an evolutionary conserved heterodimer essential for proper mRNA export and subsequently the survival of the cell. Mex67 have been studied for many years, however due to the complexity and interconnectivity of the different processes in mRNA biogenesis, there is yet to uncover many details on the dynamics of the process and the crosstalk between Mex67 and its many partners. In this study, using a combination of biochemical, biophysical, and structural analysis, we characterize the interaction between Mex67 and a novel partner protein called Mip6 (Mex67 interacting protein 6). We were able to reconstitute a stable complex in vitro, and extensively study the mechanism in which the two proteins interact. We also solved the crystal structure of the C-terminal region of Mex67 that interacts with Mip6 and identified the UBA domain of Mex67, known to bind FG nucleoporins and Hpr1 protein as also the site where Mip6 binds. However, little was known about the structure or function of Mip6 and its paralogue Pes4. Here we proved that Mip6 is an RNA binding protein with four RNA recognition motifs that binds RNA in vitro with high affinity. Additionally, its fourth RNA recognition motif was also the site of binding of Mex67. Furthermore, we showed that the Mex67 complex formation with Mip6 RRM4 compromises its ability to bind RNA or vice versa. We also designed a point mutation on Mip6 RRM4 that disrupts its interaction with Mex67 but not with RNA. Subsequent in vivo yeast assays led us to hypothesize a role of Mip6 as an adaptor protein for Mex67 in nuclear export especially upon stress. Additional function of Mip6 was the localization of its bound mRNA to cytoplasmic stress granules in cellular stress conditions. Moreover, the crystal structures of Mip6 RRM3, Pes4 RRM3, Pes4 RRM4, and Pes4 RRM3/4 were also solved. All RRMs adopted a canonical RRM fold with conserved RNP1 and RNP2 sequences normally involved in RNA binding, except Mip6 RRM3 that was missing the aromatic ring in RNP2. In the structure of RNA-free Pes4 RRM3/4, the tandem RRM domains were connected with a flexible disordered linker and no inter-domain contact between them. Finally, although Pes4 RRM4 was binding RNA in vitro, it did not have the ability to interact with Mex67 thus suggesting a separate evolutionary function for Mip6 and Pes4. / La exportación nuclear de ácido ribonucleico mensajero (ARNm) es un proceso complejo y esencial para una expresión correcta de los genes en todas las células eucariotas. La exportación de ARNm a través del complejo del poro nuclear depende principalmente de la interacción y coordinación de varias proteínas, que forman lo que se conoce como mRNPs (ribonucleoproteínas mensajeras), que tienen un papel dinámico e interconectado en las diferentes etapas de la biogénesis de ARNm, tales como el procesamiento del pre-ARNm, estabilidad, y exportación. Una proteína clave en este proceso es Mex67, conservada de levaduras a humanos, que es la principal exportadora de ARN mensajero y también está implicada en la exportación de ARN ribosomal. Mex67 interacciona con Mtr2 para formar un heterodímero conservado evolutivamente esencial para una exportación adecuada de ARNm y la consiguiente supervivencia de la célula. Se ha estudiado Mex67 durante muchos años, sin embargo, debido a la complejidad e interconectividad de los diferentes procesos de biogénesis de ARNm, todavía quedan por descubrir muchos detalles de la dinámica del proceso y las interacciones entre Mex67 y sus muchas proteínas asociadas. En este estudio, combinando un análisis bioquímico, biofísico y estructural, hemos caracterizado la interacción entre Mex67 y una nueva proteína asociada denominada Mip6 (proteína 6 que interacciona con Mex67). Hemos podido reconstituir un complejo estable in vitro y estudiar extensivamente el mecanismo por el cual interaccionan estas dos proteínas. También hemos resuelto la estructura cristalográfica de la región C-terminal de Mex67 que interacciona con Mip6 e identificado el dominio UBA de Mex67, conocido por unirse a nucleoporinas FG y a la proteína Hpr1, así como el sitio por el que se une Mip6. No obstante, se sabía muy poco sobre la estructura o la función de Mip6 y su parálogo Pes4. Hemos probado que Mip6 es una proteína de unión a ARN con cuatro motivos de reconocimiento de ARN que se unen a ARN in vitro con una afinidad alta. Además, su cuarto motivo de reconocimiento de ARN es también el sitio de unión a Mex67. Posteriormente, demostramos que la formación del complejo de Mex67 con el dominio RRM4 de Mip6 compromete su capacidad para unir ARN o viceversa. También diseñamos una mutación puntual en el RRM4 de Mip6 que rompe la interacción con Mex67 pero no con el ARN. Los ensayos posteriores in vivo en levaduras nos permitieron establecer una hipótesis sobre el papel de Mip6 como proteína adaptadora para Mex67 en la exportación nuclear, especialmente en condiciones de estrés. Una función adicional de Mip6 era la localización del ARNm que se unía a ella en gránulos de estrés en condiciones de estrés celular. Además, hemos resuelto las estructuras cristalográficas del RRM3 de Mip6, RRM3 de Pes4, RRM4 de Pes4 y los RRM3 y 4 de Pes4. Todos los RRMs adoptaron una conformación canónica RRM con secuencias RNP1 y RNP2 conservadas generalmente implicadas en la unión a ARN, excepto el RRM3 de Mip6 que carecía del anillo aromático en RNP2. En la estructura sin ARN de los RRM3 y 4 de Pes4, los dominios RRM tándem estaban conectados por una región flexible desordenada y no había un contacto inter-dominio entre ellos. Finalmente, aunque el RRM4 de Pes4 se unía a ARN in vitro, no presentaba la capacidad de interaccionar con Mex67 lo cual sugiere una divergencia evolutiva de la función de Mip6 y Pes4. / L¿exportació nuclear d¿àcid ribonucleic missatger (mRNA) es un procés complex i essencial per a una correcta expresió gènica en totes cèl¿lules eucariotes. L¿exportació del mRNA a través del complex del porus nuclear depén principalment de la interacció i coordinació de diverses proteïnes, que formen el que es coneix com mRNPs (ribonucleoproteïnes missatgeres), que tenen un paper dinàmic i interconnectat en les diferents etapes de la biogènesi d¿ARNm, com el processament del pre-ARNm, estabilitat, localització i exportació. Una proteïna clau en aquest procés és MEX67, conservada de llevats fins a humans, que és la principal exportadora de ARN missatger i també està implicada en l¿exportació de ARN ribosomal. Mex67 interacciona amb Mtr2 per a formar un heterodímer conservat evolutivament essencial per a una exportació adequada d¿ARNm i la consegüent supervivència de la cèl¿lula. S¿ha estudiat Mex67 durant molts anys, però degut a la complexitat i interconectivitat dels diferents processos de biogènesi d¿ARNm, encara queden per descobrir molts detalls de la dinàmica del procés i les interaccions entre Mex67 i les seues moltes proteïnes associades. En aquest estudi, combinant l¿anàlisi bioquímic, biofísic i estructural, hem caracteritzat la interacció entre Mex67 i una nova proteïna associada anomenada Mip6 (proteïna 6 que interacciona amb Mex67). Hem pogut reconstituir un complex estable in vitro i estudiar extensivament el mecanisme pel qual interaccionen estes dos proteïnes. També hem resolt l¿estructura cristal¿logràfica de la regió C-terminal de Mex67 que interacciona amb Mip6 i identificat el domini UBA de Mex67, conegut per unir-se a nucleoporines FG i a la proteïna Hpr1, així com ser el lloc pel que s¿uneix Mip6. No obstant, se sabia molt poc sobre l¿estructura o la funció de Mip6 i el seu paràleg Pes4. Hem comprobat que Mip6 es una proteïna d¿unió a ARN amb quatre motius de reconeixement d¿ARN que s¿uneixen a ARN in vitro amb una afinitat alta. A més, el seu quart motiu de reconeixement d¿ARN és també el lloc d¿unió a Mex67. Posteriorment, demostràrem que la formació del complex de Mex67 amb el domini RRM4 de Mip6 compromet la seua capacitat per a unir ARN o viceversa. També vam dissenyar una mutació puntual en el RRM4 de Mip6 que trenca la interacció amb Mex67 però no amb l¿ARN. Els assajos posteriors in vivo en llevats ens van permetre establir una hipòtesi sobre el paper de Mip6 com a proteïna adaptadora per a Mex67 en l¿exportació nuclear, especialment en condicions d¿estrès. Una funció adicional de Mip6 era la localització de l¿ARNm que s¿unia a ella en grànuls d¿estrès en condicions d¿estrès cel¿lular. A més, hem resolt les estructures cristal¿logràfiques del RRM3 de Mip6, RRM3 de Pes4, RRM4 de Pes4 i els RRM3 i 4 de Pes4. Tots els RRMs adoptaren una conformació canònica RRM amb seqüències RNP1 i RNP2 conservades generalment implicades en la unió a ARN, excepte el RRM3 de Mip6 que mancava del anell aromàtic en RNP2. En la estructura sense ARN dels RRM3 i 4 de Pes4, els dominis RRM tàndem estàven conectats per una regió flexible desordenada i no hi havia un contacte interdomini entre ells. Finalment, encara que el RRM4 de Pes4 es unia a ARN in vitro, no presentava la capacitat d¿interaccionar amb Mex67, la cual cosa sugerix una divergencia evolutiva de la funció de Mip6 y Pes4. / Mohamad, N. (2017). The RNA binding protein Mip6, a novel cellular partner of Mex67 export factor with implications in mRNA export [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90397

The function and evolution of introns have been topics of great interest since introns were discovered in the 1970s. Introns that interrupt protein-coding regions have the most obvious potential to affect coding sequences; therefore, their evolution have been studied most intensively. Splicing of introns within untranslated regions does not contribute directly to the diversity of proteins, yet ~35% of human transcripts contain introns within the 5' untranslated region (UTR). The evolution and possible functions of 5'UTR introns (5UIs) remain largely unexplored. Here we undertook a genome-wide functional analysis of 5UIs. Our main results are as follows: First, the distribution of these introns in the human genome is nonrandom. While genes with regulatory roles are enriched in having 5UIs, genes encoding proteins that are targeted to the endoplasmic reticulum and mitochondria are surprisingly depleted of these introns. Second, we offered and supported a model whereby gene encoding secretory and nuclear-encoded mitochondrial proteins share a common regulatory mechanism at the level of mRNA export, which is dependent on the absence of 5'UTR introns. Specifically, the upstream element in a given transcript, be it an intron or RNA elements near the 5' end of coding sequences (CDS), dictates the mRNA export pathway used. Finally, we discovered a strong correlation between existence of 5'UTR introns and sequence features near the 5' end of CDS. We developed an integrated machine-learning framework that can predict absence of 5UIs using solely the sequence near the 5' end of CDS. Our model achieved >80% accuracy when validated against nuclear-encoded mitochondrial transcripts. Specific RNA elements predictive of 5UI absence are found in ~40% of human transcripts spanning a wide spectrum of functions. By analyzing hundreds of large-scale datasets, we functionally characterized the transcripts with these RNA elements; revealing their association with translational regulation. These RNA elements were bound by proteins interacting with the Exon Junction Complex in vivo suggesting a molecular mechanism that links these elements to their downstream effects in mRNA export and translational regulation. While some 5'UTR introns might be evolving neutrally, our results, taken together, suggest that complex evolutionary forces are acting on this distinct class of introns.

Protein kinases comprise a fundamental class of cell function regulators that modify proteins by transferring phosphate groups from a nucleoside triphosphate such as ATP to specific amino acid residues on target proteins, altering protein conformation, function, and activity. As such, protein kinases are major regulators of many biological processes, including gene expression, which consists of the transfer of hereditary information in two major processing steps, transcription of DNA into a complementary precursor RNA transcript (pre-mRNA) and its subsequent translated into protein by the ribosome, where it can then go on to perform various processes in the cell. One particular family of protein kinases, otherwise known as serine/arginine protein-specific protein kinases (SRPKs), is conserved throughout eukaryotes and has been shown to be important in regulating gene expression, yet their roles in the gene expression pathway have yet to be elucidated. SRPK are known to phosphorylate serine/arginine (SR) splicing factor proteins, which are involved in mRNA splice site recognition and recruitment of splicing machinery. Members of the LAMMER kinase subfamily of SRPKs have also been shown to be required for efficient pre-mRNA splicing and important for mediating cellular progression through the cell cycle. To determine what other roles SRPKs play in mRNA processing, it is of use to study the homologous SRPK and LAMMER kinases in fission yeast, S. pombe, Dsk1 and Kic1, respectively. S. pombe provides a genetically valuable model for studying kinase function in RNA processing as both RNA processing machinery and SRPKs are conserved through higher eukaryotes. Using a novel green fluorescent protein tagging system based on properties of the MS2 bacteriophage genome, we are able to label specific mRNA transcripts of interest and visualize their locations in the cell using fluorescence microscopy. By visualizing the mRNA trafficking patterns of intron-containing and intronless mRNA transcripts, we show for the first time that deletions of the Dsk1 and Kic1 genes result in the nuclear retention of mRNA, such that Dsk1 and Kic1 are distinctly involved in mRNA export out of the nucleus.

abstract: Exome sequencing was used to identify novel variants linked to amyotrophic lateral sclerosis (ALS), in a family without mutations in genes previously linked to ALS. A F115C mutation in the gene MATR3 was identified, and further examination of other ALS kindreds identified an additional three mutations in MATR3; S85C, P154S and T622A. Matrin 3 is an RNA/DNA binding protein as well as part of the nuclear matrix. Matrin 3 interacts with TDP-43, a protein that is both mutated in some forms of ALS, and found in pathological inclusions in most ALS patients. Matrin 3 pathology, including mislocalization and rare cytoplasmic inclusions, was identified in spinal cord tissue from a patient carrying a mutation in Matrin 3, as well as sporadic ALS patients. In an effort to determine the mechanism of Matrin 3 linked ALS, the protein interactome of wild-type and ALS-linked MATR3 mutations was examined. Immunoprecipitation followed by mass spectrometry experiments were performed using NSC-34 cells expressing human wild-type or mutant Matrin 3. Gene ontology analysis identified a novel role for Matrin 3 in mRNA transport centered on proteins in the TRanscription and EXport (TREX) complex, known to function in mRNA biogenesis and nuclear export. ALS-linked mutations in Matrin 3 led to its re-distribution within the nucleus, decreased co-localization with endogenous Matrin 3 and increased co-localization with specific TREX components. Expression of disease-causing Matrin 3 mutations led to nuclear mRNA export defects of both global mRNA and more specifically the mRNA of TDP-43 and FUS. Our findings identify ALS-causing mutations in the gene MATR3, as well as a potential pathogenic mechanism attributable to MATR3 mutations and further link cellular transport defects to ALS. / Dissertation/Thesis / Doctoral Dissertation Neuroscience 2018

Les ARN messagers (ARNm) fonctionnels sont produits au cours d'un mécanisme complexe qui allie la transcription, qui permet la synthèse d'un pré-ARNm, la maturation de ce transcrit et son export. De plus, ces différentes machineries vont devoir faire face à la structure compacte de la chromatine, nécessitant une activité de décondensation/recondensation de la chromatine qui est notamment régulée par les mécanismes épigénétiques. Un très grand nombre de facteurs sont donc requis pour la production des ARNm fonctionnels . Parmi ces facteurs, les protéines Spt6 et Iws1 sont impliquées dans le mécanisme général de la transcription, dans la modulation de la structure de la chromatine et la maturation et l'export des ARNm. Ces travaux de thèse ont permis de caractériser biochimiquement, structuralement et fonctionnellement ces deux protéines, leur complexe et leur interaction avec d'autres effecteurs de la transcription. Ces travaux ont notamment permis de comprendre en termes moléculaires et fonctionnels (i) comment Spt6 est recrutée par l'ARN polyméraseII au cours de la transcription et (ii) comment le complexe Spt6/Iws 1 est formé. Ils ont également permis d'identifier de nouveaux interactants potentiels de Spt6, et notamment le facteur d'élongation de la transcription TFIIS. Ces travaux ont ainsi permis de révéler le rôle essentiel et extrêmement complexe joué par Spt6 et Iws1 lors de la production d'un ARNm, mais également de permettre l'étude future de leur interaction avec d'autres facteurs transcriptionnels.