Understanding the molecular structure of DNA was considered as greatest achievement in modern biology. It helped in understanding fundamental cellular processes such as replication of DNA, nature of the genetic code and transcription. It also led to technological advancements such as DNA sequencing, genetic engineering and gene cloning. The DNA molecule is highly polymorphic in nature and its structure is dependent on environment, base composition and sequence context. B-DNA, A-DNA, Z-DNA and curved or kinked DNA are some of the well characterized double helical polymorphs. B-DNA is the most prevalent structure in vivo and it can undergo small local variations and global variations. In this thesis we refer to distinct structural property of any particular DNA sequence as deviation from fibre model B-DNA structural parameters or random sequence DNA. Structural properties of DNA are an outcome of the linear arrangement of the 4 chemically different nucleotide bases and the characteristic features of the two grooves (minor and major) arising due to the asymmetric position of glycosidic bonds of base pairs. DNA structure and properties are expected to vary along its length. Several structural features have been defined for DNA duplex, while DNA stability, bendability and intrinsic curvature are well studied and found to be biologically relevant. These three sequence dependent properties differ in their nature and information content and can be studied both at local and global levels, depending on the length of DNA fragment being examined. Majority of the work in this thesis focuses on the analysis of these three DNA structural features in promoter regions of different eukaryotic systems and their relationship with gene expression. The thesis work is divided in to five sections briefly described below. The sections discuss prevalence of the three structural features, DNA stability, bendability and intrinsic curvature in the promoter regions of six eukaryotic systems namely S. cerevisiae, D. melanogaster, C. elegans, zebrafish, mouse and human. The relationship between DNA structural features of promoter regions of S. cerevisiae with gene expression variability is discussed, followed by application of the structure-based promoter prediction algorithm ‘PromPredict’ in annotating promoter regions of six different eukaryotes. Finally, an analysis of structural features of the flanking sequences of transcription factor binding sites (TFBSs) of six transcription factors and their relationship with the DNA binding affinity is discussed. Each of the projects described below will appear as a separate chapters in the thesis. An overview of the eukaryotic transcription machinery, promoter elements and different DNA structural properties are discussed in the introduction of the thesis (chapter 1). The structural properties of DNA in the promoter regions of eukaryotic genes (chapter 2)Earlier studies in the lab reported that, apart from sequence motifs, promoter re- gions have distinct structural properties, such as lower stability, lesser bendability and more curvature compared to other genomic regions. But those studies were on small datasets and few model systems. Advancement in high-throughput tech- niques has made availability of transcription start site information for many model systems. This work was initiated with the aim of investigating the structural fea- tures in different eukaryotic systems belonging to different domains of life. The quantitative analysis of three different structural features of promoter regions of six different model systems S. cerevisiae, C. elegans, D. melanogaster, zebrafish, mouse and human has been carried out. Further, the composition of different k-mers (k=3, 4 and 6) A-tracts and G-quadruplexes has been studied. The analysis allowed us to understand the similarities and differences in struc- tural features of promoter sequences in different model systems. The core promoter sequences of S. cerevisiae, C. elegans, D. melanogaster, zebra fish, mouse and hu- man have been observed to be less stable and have lower preference for nucleosome formation. S. cerevisiae, C. elegans and D. melanogaster promoter sequences have been shown to be less bendable whereas zebrafish, mouse and human promoter se- quences are flexible in terms of bendability towards major groove as predicted fDNase 1 sensitivity model. S. cerevisiae, C. elegans, D. melanogaster core promoter regions have AT rich oligomers, whereas mouse and human core promoter regions have GC rich oligomers and G-quadruplex motifs. DNA structural features of TATA-containing andTATA-less promoters (chapter 3)Eukaryotic genes can be broadly classified as TATA-containing and TATA-less based on the presence of TATA-box in their promoter sequences. Experiments on both classes of genes have reported that, they have differences in regulation of gene ex- pression and cellular functions. In this chapter, the differences in compositional and structural features of TATA-containing and TATA-less promoters in the above mentioned model systems are discussed. The results suggested that DNA structural features of TATA-containing and TATA-less promoters are distinctly different in all eukaryotes. The TATA-containing promoters are less stable, more flexible and more curved compared to TATA-less promoters in lower eukaryotes. In mouse and hu- man genes, DNA duplex stability and G-quadruplex motifs are very distinguishing features in the two classes of promoters. DNA structural properties of eukaryotic promoter regions and gene expression variability (chapter 4) Gene expression is regulated by various external (environment and evolution) and internal (genetic) factors. Presence of sequence motifs, such as TFBSs and TATA- box, as well as DNA methylation has been implicated in the regulation of expression of some genes in vertebrates, but a large number of genes lack these sequences. Ear- lier analyses (described in previous sections) in S. cerevisiae, have shown that their promoter sequences have special structural properties, such as low stability, less bendability and more curvature compared to other genomic regions. These strutural features may play a role in transcription initiation and regulation of gene expression. This project was carried out to understand 1. What is the relationship between DNA structural features and gene expres- sion? 2. What is the relationship between gene expression and bidirectionality of a pro- moter region? For this purpose, the information of seven different gene expression variability measures, stochastic noise, responsiveness, stress response, trans variability, mu- tational variance, interstrain variation and expression divergence have been com- pared with structural features in the promoter regions. It is observed that a few of the variability measures of gene expression are linked to DNA structural prop- erties, along with nucleosome occupancy, TATA-box presence and bidirectionality of promoter regions. Interestingly, gene responsiveness is shown to be most, inti- mately correlated with DNA structural features and promoter architecture. The study highlights the importance of sequence dependent structural features in gene regulation. Promoter prediction in eukaryotes using DNA duplex stability (chapter 5) Structural property-based algorithms can discriminate promoter sequences from non-promoter sequences and are far better than sequence motif-based predictors. Compared to other structural features, low stability is found to be the most preva- lent feature in promoter regions. “PromPredict” (in-house algorithm) uses the din- ucleotide free energy values obtained from differential melting stability of DNA du- plexes as a predictor of promoters and has been successfully used earlier to annotate promoter sequences in prokaryotes and rice. Comprehensive analysis of the perfor- mance of PromPredict in S. cerevisiae, D. melanogaster, C. elegans, zebrafish, mouse and human as well as TATA-containing and TATA-less promoter regions of S. cere visiae with TSS data and 48 eukaryotic systems with translation start site (TLS) data revealed that differential stability is a good criterion for promoter prediction. DNA structure in flanking sequences of consensus motifs modulate transcription factor binding (chapter 6) Sequence specific DNA-protein interactions are essential for specific expression pat- terns during the development. There are several factors contribute to DNA-binding specificities of transcription factors (TFs). They include structure and flexibility of TFs, cofactors, chromatin environment and DNA sequence. Along with actual tran- scription factor binding sites (TFBSs), their sequence context (flanking sequences) is also shown to play a major role in gene regulation. Most of the studies have ad- dressed the sequence context at global level but very little is understood about the role of sequences flanking TFBSs in binding of transcription factors. This project was initiated with the aim of understanding the effect of flanking sequences of TFBSs in transcription factor binding affinity. In vitro DNA binding information of six different transcription factors (with three types of DNA bind- ing domains, Zinc finger (GATA4), home domain (AbdA, AbdB and Ubx) and bZIP (fos-jun and Nfil3)) was provided by Aseem Ansari’s lab. The compositional and structural features (minor groove width, propeller twist, wedge and free energy) are compared with the DNA binding profiles of 12mers (or 8mers) of six different transcription factors. It has been observed that some of the DNA structural proper- ties of flanking sequences are strongly correlated with binding affinity. For GATA4 sequences, binding affinity is negatively correlated to GC content or minor groove width at their 5′ -flanking region, showing the significance of narrow minor groove at 5′ -region. On the other hand, the binding affinity of bZIP proteins is negatively correlated to wedge angles, whereas in case of homeodomain proteins, it is posi- tively correlated to propeller twist and GC content. Thus, this study highlights the differential preference for flanking sequences outside the core binding motifs of six different TFs, which interact with DNA through α-helix. ‘The relationship between transcription pre-initiation complexes and gene ex- pression variability in S. cerevisiae’ is briefly described in the appendix section of the thesis. General conclusion Overall, the results presented in this thesis indicate that DNA sequence based structural features are unique to promoter regions and play an important role in gene regulation. Local structural features of flanking sequences of transcription factor binding sites are also instrumental in determining the DNA binding affinity of transcription factors.

No description available.

Hepatitis delta virus (HDV) is the smallest known mammalian RNA virus, containing a genome of ~ 1700 nt. Replication of HDV is extremely dependent on the host transcription machinery. Previous studies indicated that RNA polymerase II (RNAPII) directly binds to and forms an active preinitiation complex on the right terminal stem-loop fragment (R199G) of HDV genomic RNA, and that the polypyrimidine tract-binding protein-associated splicing factor (PSF) directly binds to the same region. Further studies demonstrated that PSF also binds to the carboxyl-terminal domain (CTD) of RNAP II. In my thesis, co-immunoprecipitation assays were performed to show that PSF stimulates the interaction of RNAPII with R199G. Results of co-immunoprecipitation experiments also suggest that both the RNA recognition motif 2 (RRM2) and N-terminal proline-rich region (PRR) of PSF are required for the interaction between PSF and RNAPII, while the two RNA recognition motifs (RRM1 and RRM2) might be required for the interaction of PSF with R199G. Furthermore, in vitro run-off transcription assays suggest that PSF facilitates the HDV RNA transcription from the R199G template. Together, the above experiments suggest that PSF might act as a transcription factor for the RNAPII transcription of HDV RNA by linking the CTD of RNAPII and the HDV RNA promoter. My experiments provide a better understanding of the mechanism of HDV RNA-dependent transcription by RNAP II.

The innate immune system is a multicomponent response governed by intricate mechanisms of induction, regulation and resolution to elicit antimicrobial defenses. In recent years, the complexity of eukaryotic transcriptomes has become the subject of intense scrutiny and curiosity. It has been established, that RNA polymerase II (RNAPII) transcribes hundreds to thousands of long noncoding RNAs (lncRNAs), often in a stimulus and cell-type specific manner. However, the functional significance of these transcripts has been particularly controversial. While the number of identified lncRNAs is growing, our understanding of how lncRNAs themselves regulate other genes is quite limited. In chapter 2, a novel lncRNA is identified, more specifically, a natural antisense transcript, that mediates the transcription of the pro-inflammatory cytokine IL-1α. Through loss-of-function studies, I report the necessity of this transcript in mediating IL-1α mRNA expression by affecting RNAPII binding to the IL-1α promoter after toll-like receptor signaling. For the first time, I show that IL-1α is regulated at the transcriptional level. As a second independent component of this thesis, we explore the role of the innate immune response after infection by the malaria-causing parasite, Plasmodium berghei ANKA (PbA), and how innate immune components are both beneficial and detrimental to the host depending on when and where inflammation is triggered during infection. We attempt to identify the “malarial toxin” responsible for aberrations in the immune response that is detrimental for disease outcomes and the innate signaling pathways that are involved. Many pathogens induce pathological inflammatory conditions that lead to irreparable homeostatic imbalances and become fatal to the host. Here, type I Interferon signaling is required to dampen parasite load during liver-stage infections, but leads to host mobidity if these pathways are activated in the erythrocytic phase of infection. Together, this thesis provides new insights on how components of the innate immune system are regulated, and how dysregulation of immunity can potentially lead to adverse effects during active infections.

Indiana University-Purdue University Indianapolis (IUPUI) / The C-terminal domain (CTD) of the RNA polymerase II (RNAPII) subunit Rpb1 must exist in a hypophosphorylated state prior to forming a competent transcription initiation complex. However, during transcription, specific kinases and phosphatases act on the RNAPII CTD to regulate its phosphorylation state, which serves to recruit sequence-specific and general transcription factors at the appropriate stage of transcription. A key phosphatase involved in this process, Rtr1 (Regulator of Transcription 1), was shown to regulate a key step important for transcription elongation and termination. Although the role that Rtr1 plays in regulating RNAPII transcription has been described, the mechanism involved in the recruitment of Rtr1 to RNAPII during transcription has not been elucidated in yeast. Consequently, the present work utilized both affinity purification schemes in Saccharomyces cerevisiae and mass spectrometry to identify key Rtr1-interacting proteins and post-translational modifications that potentially play a role in recruiting Rtr1 to RNAPII. In addition to RNAPII subunits, which were the most consistently enriched Rtr1-interacting proteins, seven proteins were identified that are potentially involved in Rtr1 recruitment. These included PAF complex subunits (Cdc73, Ctr9, Leo1), the heat shock protein Hsc82, the GTPase Npa3, the ATPase Rpt6, and Spn1. Indirect evidence was also uncovered that implicates that the CTDK-I complex, a kinase involved in RNAPII CTD phosphorylation, is important in facilitating interactions between Rtr1, RNAPII, and select transcription factors. Additionally, a putative phosphorylation site was identified on Ser217 of Rtr1 that may also play a role in its recruitment to RNAPII during transcription.

Le transport et la localisation des ARN messagers permettent de réguler l’expression spatiale et temporelle de facteurs spécifiques impliqués dans la détermination du destin cellulaire, la plasticité synaptique, la polarité cellulaire et la division asymétrique des cellules. Chez S.cerevisiæ, plus de trente transcrits sont transportés activement vers le bourgeon cellulaire. Parmi ces transcrits, l’ARNm ASH1 (asymetric synthesis of HO) est localisé à l’extrémité du bourgeon pendant l’anaphase. Ce processus va entrainer une localisation asymétrique de la protéine Ash1p, qui sera importée uniquement dans le noyau de la cellule fille, où elle entraine le changement de type sexuel. La localisation asymétrique de l’ARNm ASH1, et donc de Ash1p, implique la présence de différents facteurs de localisation. Parmi ces facteurs, les protéines She (She1p/Myo4p, She2p et She3p) et les répresseurs traductionnels (Puf6p, Loc1p et Khd1p) participent à ce mécanisme. La protéine navette She2p est capable de lier l’ARNm ASH1 et va entrainer le ciblage de cet ARNm vers l’extrémité du bourgeon en recrutant le complexe She3p-Myo4p. Des répresseurs traductionnels régulent la traduction de cet ARNm et évitent l’expression ectopique de la protéine Ash1p pendant son transport. Alors que la fonction cytoplasmique de She2p sur la localisation des ARNm est connue, sa fonction nucléaire est encore inconnue. Nous avons montré que She2p contient une séquence de localisation nucléaire non classique qui est essentielle à son import nucléaire médié par l’importine α (Srp1p). L’exclusion de She2p du noyau par mutation de son NLS empêche la liaison de Loc1p et Puf6p sur l’ARNm ASH1, entrainant un défaut de localisation de l’ARNm et de la protéine. Pour étudier plus en détail l’assemblage de la machinerie de localisation des ARNm dans le noyau, nous avons utilisé des techniques d’immunoprécipitation de chromatine afin de suivre le recrutement des facteurs de localisation et des répresseurs traductionnels sur les ARNm naissants. Nous avons montré que She2p est recruté sur le gène ASH1 pendant sa transcription, via son interaction avec l’ARNm ASH1 naissant. Puf6p est également recruté sur ASH1, mais d’une manière dépendante de la présence de She2p. De façon intéressante, nous avons détecté une interaction entre She2p et la plus grande sous-unité de l’ARN polymérase II (Rpb1p). Cette interaction est détectée avec la forme active en élongation de l’ARN polymérase II. Nous avons également démontré que She2p interagit avec le complexe d’élongation de la transcription Spt4p/Spt5p. Une délétion de SPT4 ou une mutation dans SPT5 (Ts spt5) à température restrictive empêche l’interaction entre She2p et Rpb1p, et diminue le recrutement de She2p au gène ASH1, entrainant un défaut de localisation de l’ARNm et un défaut de localisation asymétrique de la protéine Ash1p. De manière globale, nos résultats montrent que les facteurs impliqués dans la localisation cytoplasmique des ARNm et dans leur contrôle traductionnel sont recrutés de façon co-transcriptionnelle sur les ARNm naissants via leur interaction avec la machinerie de transcription, suggèrant un rôle important de la machinerie transcriptionelle dans la localisation des ARNm. / Cytoplasmic transport and localization of messenger RNAs allows temporal and spatial expression of specific factors involved in cell fate determination, synaptic plasticity, cellular polarity or asymmetric cell division. In S. cerevisiae, over thirty transcripts are actively transported and localized to the bud tip of budding yeast. One of them, the ASH1 mRNA (for Asymmetric Synthesis of HO), is localized at the bud tip in late anaphase cells. This allows Ash1p, a transcriptional repressor of the HO endonuclease, to be sorted exclusively to the daughter cell nucleus, where it prevents mating type switching. Proper ASH1 mRNA localization and Ash1p asymmetric expression involve localization factors, which are part of the She-proteins (She1p/Myo4p, She2p and She3p), and translational repressors (the proteins Puf6, Loc1 and Khd1). The nucleo-cytoplasmic shuttling protein She2p binds the ASH1 mRNA and targets it for localization at the bud tip by recruiting the She3p-Myo4p complex. Translational repressors regulate the translation of ASH1 mRNA and avoid ectopic expression of the Ash1 protein during the transport of its transcript. While the cytoplasmic role of She2p in mRNA localization is known, its nuclear function is still unclear. We now show that She2p contains a non-classical nuclear localization signal sequence (NLS) which is essential for its nuclear import via the importin  Srp1p. Exclusion of She2p from the nucleus by mutagenesis of its NLS disrupt the binding of Loc1p and Puf6p to the ASH1 mRNA, leading to defective mRNA localization and Ash1p sorting. To further investigate the assembly of the mRNA localization machinery in the nucleus, we used chromatin immunoprecipitation (ChIP) to follow the recruitment of localization factors and translational repressors on nascent localized mRNAs. We found that She2p is recruited on the ASH1 gene during transcription, via its interaction with the nascent ASH1 mRNA. Puf6p is also recruited on the ASH1 gene, but in a She2p-dependent manner. Interestingly, we detected an interaction between She2p and Rpb1p, the largest subunit of RNA polymerase II in vivo. This interaction is independent of the RNA-binding properties of She2p, and involves the elongating form of the RNA polymerase II. We also found that She2p interacts with both members of the elongation factors Spt4p /Spt5p; Deletion of SPT4 or Ts spt5 mutants at restrictive temperature disrupted the interaction between She2p and Rpb1p, and then reduced the recruitment of She2p on the ASH1 gene, resulting in ASH1 mRNA delocalization and defective Ash1p sorting. Altogether, our results show that factors involved in cytoplasmic mRNA localization and translational control are recruited co-transcriptionally on nascent mRNAs via interation with the transcription machinery, pointing toward a role of the transcription machinery in the mRNA localization process.

PolyHistidine repeats and DYRK1A: from the localization to the functionEl principal objectiu d'aquesta tesi ha estat el d'esbrinar noves funcions de la proteína quinasa DYRK1A en el nucli cel.lular. Donat que el domini de repetició d'histidines de DYRK1A dirigeix la proteína al compartiment d'speckles nuclears, aquesta propietat ha estat utilitzada per adreçar aquesta pregunta. Els resultats obtinguts en aquesta tesi han permès proposar els homopolímers d'histidina com una nova i general senyal de localització a speckles nuclears. Proteïnes amb segments de polihistidines, la majoria d'elles factors de transcripció, mostren un comportament intranuclear dinàmic, compatible amb un model en el quèl diferents dominis d'interacció competeixen entre ells pel reclutament de la proteína a diferents subcompartiments nuclears. El mecanisme molecular que media l'acumulació a speckles de les proteïnes amb polihistines s'ha estudiat utilitzant DYRK1A com a model. Els resultats obtinguts exclouen la unió a l'RNA com a mecanisme de reclutament i concloure que, aquest, ocorre mitjançant la interacció amb proteïnes residents. S'han identificat dues noves proteïnes interactores per a DYRK1A, l'RNA polimerasa II i el factor de transcripció Brn-3b. La fosforilació de DYRK1A sobre el domini C-terminal o CTD de l'RNA polimerasa II suggereix una funció directa de la quinasa en el procés de transcripció o del seu acoblament al processament d'RNAs missatgers. La fosforilació de DYRK1A sobre el domini d'activació de Brn-3b sembla regular positivament l'activitat transcripcional d'aquest factor. Aquests resultats indiquen una funció activa de DYRK1A en la regulació de la transcripció gènica, tant directament sobre la maquinària transcripcional com indirectament, modulant l'activitat de factors de transcripció. PolyHistidine repeats and DYRK1A: from the localization to the functionThe main objective of this thesis work has been to identify new roles for the protein kinase DYRK1A in the cell nucleus. Given that a histidine repeat in DYRK1A targets the protein to the nuclear speckle compartment, this property has been used as a tool to approach the question. The results obtained in this thesis work have allowed proposing homopolymeric histidine runs as a novel and general nuclear speckle-directing signal. Proteins with polyHistidine segments, mostly transcription factors, present a dynamic intranuclear behaviour compatible with a model in which distinct interacting domains compete for recruiting elements within the nucleus. The molecular mechanisms that mediate speckle accumulation have been studied in DYRK1A as a model system. The results allow excluding RNA binding as the recruiting mechanism and concluding that targeting is mediated by interaction with speckle-resident proteins. Two novel DYRK1A interactors have been identified during the study, the RNA polymerase II and the transcription factor Brn-3b. DYRK1A phosphorylation of the C-terminal domain or CTD of the RNA polymerase II suggests a direct role of DYRK1A on transcription or coupling of transcription with RNA processing. DYRK1A phosphorylation of Brn-3b within its activation domain seems to positively regulate Brn-3b transcriptional activity. These results confirm an active role for DYRK1A in gene transcription regulation both direct on the transcriptional machinery and indirect by modulating the activity of transcription factors.

En el llevat Saccharomyces cerevisiae els canvis en les condicions osmòtiques del medi extracel.lular són sensades per la MAP cinasa Hog1, la qual permet dur a terme l'adaptació cel.lular mitjançant la modulació de l'expressió gènica, de la traducció i de la progressió del cicle cel.lular. A l'inici d'aquest projecte de tesi, els mecanismes pels quals Hog1 controla l'expressió gènica no eren del tot coneguts. El nostre objectiu va ser caracteritzar el mecanisme molecular pel qual Hog1 modula la transcripció en resposta a estrès osmòtic. Hem aconseguit demostrar que el reclutament de Hog1 als promotors sensibles a estrès osmòtic per part del factor de transcripció és essencial per al reclutament i activació de la RNA polimerasa II, mecanisme que podria estar conservat en les cèl.lules eucariotes. També hem identificat noves activitats remodeladores de cromatina implicades en la resposta gènica a osmoestrès mediada per Hog1. Vàrem realitzar un cribatge genètic per identificar mutacions que provoquessin osmosensibilitat i una reducció en l'expressió de gens de resposta a estrès osmòtic. Aquest cribatge ens va permetre identificar nous reguladors de la transcripció mediada per osmoestrès: la histona deacetilasa Rpd3 i els complexes SAGA i mediador. Els nostres resultats permeten, doncs, definir un important paper per a Rpd3, SAGA i mediador en la inducció gènica mediada per Hog1, i han estat importants per assolir una millor visió de com les cinases activades per estrès regulen la iniciació de la transcripció. / In Saccharomyces cerevisiae, changes in the extracellular osmotic conditions are sensed by the HOG MAPK pathway, which elicits the program for cell adaptation, including modulation of gene expression, translation and cell-cycle progression. At the beginning of this PhD Project, the mechanisms by which Hog1 was controlling gene transcription were not completely understood. Our main objective was to characterize the molecular mechanisms by which the Hog1 MAPK modulates transcription upon osmostress. We have shown that anchoring of Hog1 to osmoresponsive promoters by the transcription factor is essential for recruitment and activation of RNA polymerase II, a mechanism that might be conserved among eukaryotic cells. In addition, we identified novel chromatin modifying and remodelling activities involved in the Hog1-mediated osmostress gene expression. We performed a genome-wide genetic screening searching for mutations that render cells osmosensitive and displayed reduced expression of osmoresponsive genes. Rpd3 histone deacetylase, SAGA and Mediator complexes were identified as novel regulators of osmostress-mediated transcription. Thus, our results define a major role for Rpd3, SAGA and Mediator in the Hog1-mediated osmostress gene induction, and have been important to achieve a better view of how a SAPK regulates transcription initiation.

Le transport et la localisation des ARN messagers permettent de réguler l’expression spatiale et temporelle de facteurs spécifiques impliqués dans la détermination du destin cellulaire, la plasticité synaptique, la polarité cellulaire et la division asymétrique des cellules. Chez S.cerevisiæ, plus de trente transcrits sont transportés activement vers le bourgeon cellulaire. Parmi ces transcrits, l’ARNm ASH1 (asymetric synthesis of HO) est localisé à l’extrémité du bourgeon pendant l’anaphase. Ce processus va entrainer une localisation asymétrique de la protéine Ash1p, qui sera importée uniquement dans le noyau de la cellule fille, où elle entraine le changement de type sexuel. La localisation asymétrique de l’ARNm ASH1, et donc de Ash1p, implique la présence de différents facteurs de localisation. Parmi ces facteurs, les protéines She (She1p/Myo4p, She2p et She3p) et les répresseurs traductionnels (Puf6p, Loc1p et Khd1p) participent à ce mécanisme. La protéine navette She2p est capable de lier l’ARNm ASH1 et va entrainer le ciblage de cet ARNm vers l’extrémité du bourgeon en recrutant le complexe She3p-Myo4p. Des répresseurs traductionnels régulent la traduction de cet ARNm et évitent l’expression ectopique de la protéine Ash1p pendant son transport. Alors que la fonction cytoplasmique de She2p sur la localisation des ARNm est connue, sa fonction nucléaire est encore inconnue. Nous avons montré que She2p contient une séquence de localisation nucléaire non classique qui est essentielle à son import nucléaire médié par l’importine α (Srp1p). L’exclusion de She2p du noyau par mutation de son NLS empêche la liaison de Loc1p et Puf6p sur l’ARNm ASH1, entrainant un défaut de localisation de l’ARNm et de la protéine. Pour étudier plus en détail l’assemblage de la machinerie de localisation des ARNm dans le noyau, nous avons utilisé des techniques d’immunoprécipitation de chromatine afin de suivre le recrutement des facteurs de localisation et des répresseurs traductionnels sur les ARNm naissants. Nous avons montré que She2p est recruté sur le gène ASH1 pendant sa transcription, via son interaction avec l’ARNm ASH1 naissant. Puf6p est également recruté sur ASH1, mais d’une manière dépendante de la présence de She2p. De façon intéressante, nous avons détecté une interaction entre She2p et la plus grande sous-unité de l’ARN polymérase II (Rpb1p). Cette interaction est détectée avec la forme active en élongation de l’ARN polymérase II. Nous avons également démontré que She2p interagit avec le complexe d’élongation de la transcription Spt4p/Spt5p. Une délétion de SPT4 ou une mutation dans SPT5 (Ts spt5) à température restrictive empêche l’interaction entre She2p et Rpb1p, et diminue le recrutement de She2p au gène ASH1, entrainant un défaut de localisation de l’ARNm et un défaut de localisation asymétrique de la protéine Ash1p. De manière globale, nos résultats montrent que les facteurs impliqués dans la localisation cytoplasmique des ARNm et dans leur contrôle traductionnel sont recrutés de façon co-transcriptionnelle sur les ARNm naissants via leur interaction avec la machinerie de transcription, suggèrant un rôle important de la machinerie transcriptionelle dans la localisation des ARNm. / Cytoplasmic transport and localization of messenger RNAs allows temporal and spatial expression of specific factors involved in cell fate determination, synaptic plasticity, cellular polarity or asymmetric cell division. In S. cerevisiae, over thirty transcripts are actively transported and localized to the bud tip of budding yeast. One of them, the ASH1 mRNA (for Asymmetric Synthesis of HO), is localized at the bud tip in late anaphase cells. This allows Ash1p, a transcriptional repressor of the HO endonuclease, to be sorted exclusively to the daughter cell nucleus, where it prevents mating type switching. Proper ASH1 mRNA localization and Ash1p asymmetric expression involve localization factors, which are part of the She-proteins (She1p/Myo4p, She2p and She3p), and translational repressors (the proteins Puf6, Loc1 and Khd1). The nucleo-cytoplasmic shuttling protein She2p binds the ASH1 mRNA and targets it for localization at the bud tip by recruiting the She3p-Myo4p complex. Translational repressors regulate the translation of ASH1 mRNA and avoid ectopic expression of the Ash1 protein during the transport of its transcript. While the cytoplasmic role of She2p in mRNA localization is known, its nuclear function is still unclear. We now show that She2p contains a non-classical nuclear localization signal sequence (NLS) which is essential for its nuclear import via the importin  Srp1p. Exclusion of She2p from the nucleus by mutagenesis of its NLS disrupt the binding of Loc1p and Puf6p to the ASH1 mRNA, leading to defective mRNA localization and Ash1p sorting. To further investigate the assembly of the mRNA localization machinery in the nucleus, we used chromatin immunoprecipitation (ChIP) to follow the recruitment of localization factors and translational repressors on nascent localized mRNAs. We found that She2p is recruited on the ASH1 gene during transcription, via its interaction with the nascent ASH1 mRNA. Puf6p is also recruited on the ASH1 gene, but in a She2p-dependent manner. Interestingly, we detected an interaction between She2p and Rpb1p, the largest subunit of RNA polymerase II in vivo. This interaction is independent of the RNA-binding properties of She2p, and involves the elongating form of the RNA polymerase II. We also found that She2p interacts with both members of the elongation factors Spt4p /Spt5p; Deletion of SPT4 or Ts spt5 mutants at restrictive temperature disrupted the interaction between She2p and Rpb1p, and then reduced the recruitment of She2p on the ASH1 gene, resulting in ASH1 mRNA delocalization and defective Ash1p sorting. Altogether, our results show that factors involved in cytoplasmic mRNA localization and translational control are recruited co-transcriptionally on nascent mRNAs via interation with the transcription machinery, pointing toward a role of the transcription machinery in the mRNA localization process.

Indiana University-Purdue University Indianapolis (IUPUI) / RNA polymerase II (RNAPII) is regulated by multiple modifications to the C-terminal domain (CTD) of the largest subunit, Rpb1. This study has focused on the relationship between hyperphosphorylation of the CTD and RNAPII turnover and proteolytic degradation as well as post-translational modifications of the globular core of RNAPII. Following tandem affinity purification, western blot analysis showed that MG132 treated RTR1 ERG6 deletion yeast cells have accumulation of total RNAPII and in particular, the hyperphosphorylated form of the protein complex. In addition, proteomic studies using MuDPIT have revealed increased interaction between proteins of the ubiquitin-proteasome degradation system in the mutant MG132 treated yeast cells as well as potential ubiquitin and phosphorylation sites in RNAPII subunits, Rpb6 and Rpb1, respectively. A novel Rpb1 phosphorylation site, T1471-P, is located in the linker region between the CTD and globular domain of Rpb1 and will be the focus of future studies to determine biological significance of this post-translational modification.