Gene expression can be regulated at multiple steps once transcription is initiated. I have studied two different gene models, the α-Fetoprotein (AFP) and the immunoglobulin heavy chain (IgM) genes, to better understand post-transcriptional gene regulation mechanisms. The AFP gene is highly expressed during fetal liver development and dramatically repressed after birth. There is a mouse strain-specific difference between adult levels of AFP, with BALB/cJ mice expressing 10 to 20-fold higher levels compared to other mouse strains. BALB/cJ mice express low levels of Zhx2 and thus incompletely repress AFP. Despite differences in steady state AFP mRNA levels in the adult liver between Balb/cJ and wild-type mice, transcription rates across this gene were similar, indicating a post-transcriptional regulatory mechanism. I found accumulated unspliced RNA across multiple AFP introns in wild-type mice where mature AFP mRNA levels are low, suggesting overall AFP splicing is inefficient in the presence of Zhx2. The IgM gene is alternative processed to produce two mRNA isoforms through a competition between cleavage/polyadenylation (μspA) and splicing reactions and the pA/splice RNA expression ratio increases during B cell maturation. Cotranscriptional cleavage (CoTC) events, driven by specific cis-acting elements, are required downstream of some poly(A) signals to terminate transcription. In some cases, a pause site can produce similar effect. I explored whether there is a CoTC-like element within the IgM gene that may contribute to developmental changes in the mRNA ratio. In both a B cell and plasma cell line there was a gradual decrease in transcripts downstream from the μspA signal, suggesting that there is not evidence for a CoTC element within the IgM gene. To examine the effect a CoTC element would have on the competition between the splice and μspA reactions, we inserted the CoTC sequence of the β-globin gene into different locations downstream of the μspA signal. While the β-globin CoTC element caused cotranscriptional cleavage in all locations, it only affected the μspA/splice ratio when located close to the μspA site. This suggests there is a position effect of the inserted CoTC element on the competing polyadenylation and splicing reactions within the IgM transcripts.

Splicing mainly occurs co-transcriptionally, suggesting that transcription and premRNA splicing could be synchronized. The nature of this phenomenon suggests that transcription elongation rate may influence splicing outcomes and, indeed, there is evidence for effects on alternative splicing in mammals. To elucidate potential effects of transcription rate on splicing efficiency and fidelity, splicing of nascent transcripts was investigated in fast and slow elongating RNA polymerase II (RNAPII) mutants in Saccharomyces cerevisiae. High kinetic resolution 4-thio Uracil labelling of nascent RNA reveals that fast RNAPII accumulates unspliced pre-mRNA that represents reduced co-transcriptional splicing. Conversely, low levels of unspliced pre-mRNA were detected in the slow mutant due to increased co-transcriptional splicing. The highly stable association of nascent transcripts with elongating RNAPII permits co-transcriptional splicing to be measured by analysis of transcripts that co-purify with RNAPII. Measuring co-precipitation of the spliced mRNA and excised intron that are associated with RNAPII demonstrates that splicing is mostly co-transcriptional with the slow mutant, and the fast mutant reduces co-transcriptional splicing. How elongation rate affects splicing fidelity in budding yeast and whether faster and slower transcription have the opposite effect on splicing fidelity as might be predicted by the kinetic coupling model is an open question. Using deep RNA sequencing, splicing fidelity was determined in yeast transcription elongation mutants. Results show that both fast and slow transcription reduce splicing fidelity mainly in ribosomal protein coding transcripts. Analysis reveals that splicing fidelity depends largely on intron length, secondary structure and splice site score. These analyses also provide new insights regarding the effect of altering transcription rate on selection of transcription start sites. Together, these results indicate that optimal splicing efficiency and fidelity require finely-tuned transcription speed.

L’Splicing de les molècules d’ARN és el procés pel qual les seqüències interposades (“introns”) s’eliminen, i les seqüències restants es concatenen per a formar l’ARN madur. La investigació recent mostra que gairebé tots els gens amb splicing es veuen afectats per splicing alternatiu. Aquí, en primer lloc definim la longitud mínima d’un oligomer d’ARN per a funcionar com a lloc d’unió d’un factor d’splicing. A continuació, explorem la capacitat d’aquests oligomers per a predir estructures completes exó-intró. Destaquem els oligomers que són més informatius per a això, i demostrem que la mateixa precisió com en enfocaments anteriors es pot aconseguir amb menys oligomers. L’observació de que aquest enfocament és lluny de predir amb exactitud tota l’estructura exó-intró ens va portar a investigar els factors que juguen un paper en l’splicing co-transcripcional. Demostrem que els nucleosomes es col.loquen preferentment en els exons i plantegem la hipòtesi que juguen un paper en les decisions de l’splicing. A continuació, introduïm el “completed splicing index” i concluem que l’splicing co-transcripcional és molt generalitzat. A més, l’splicing co-transcripcional mostra vincles amb l’organització de la cromatina. A la llum d’aquests resultats, es van supervisar els canvis de la cromatina en exons diferencialment inclosos en dos teixits. Hem descobert una varietat de marques de les histones, però no totes, mostrant un comportament significativament diferent en els exons més inclosos i més exclosos. Las marques més destacades que apareixen són H3K9ac i dos estats de metilació de lisina 4. / Splicing of RNA molecules is the process, by which intervening sequences (“introns”) in the primary transcript are excised, and the remaining sequences (“exons”) are concatenated to form the mature RNA. Recent evidence shows that almost all spliced genes are affected by alternative splicing. Here, we define the minimal length of RNA oligomers that can sensibly be called splicing factor binding sites. Then, we explore the capacity of these oligomers to predict complete exon-intron structures. We highlight those oligomers that are most informative for this and show, that equal accuracy as in previous approaches can be achieved with less RNA oligomers. The observation, that this approach falls short of accurately predicting the entire exon-intron structure, led us to investigate determinants linked to co-transcriptional splicing. We show that nucleosomes are preferentially positioned on exons and hypothesize that they play a role in splicing decisions. We then introduce the “completed splicing index” and conclude that co-transcriptional splicing is very wide-spread in humans. Furthermore co-transcriptional splicing exhibits links to chromatin organization. In the light of these results, we go on to monitor chromatin changes on differentially included exons in pair-wise tissue comparisons. We find a variety of histone marks, but not all, showing significantly different behavior on up- and downregulated exons. The most prominently appearing marks are H3K9ac and two lysine 4 methylation states.

Cellular function and physiology are largely established through regulated gene expression. The first step in gene expression, transcription of the genomic DNA into RNA, is a process that is highly aligned at the levels of initiation, elongation and termination. In eukaryotes, protein-coding genes are exclusively transcribed by RNA polymerase II (Pol II). Upon transcription of the first 15-20 nucleotides (nt), the emerging nascent RNA 5’ end is modified with a 7-methylguanosyl cap. This is one of several RNA modifications and processing steps that take place during transcription, i.e. co-transcriptionally. For example, protein-coding sequences (exons) are often disrupted by non-coding sequences (introns) that are removed by RNA splicing. The two transesterification reactions required for RNA splicing are catalyzed through the action of a large macromolecular machine, the spliceosome. Several non-coding small nuclear RNAs (snRNAs) and proteins form functional spliceosomal subcomplexes, termed snRNPs. Sequentially with intron synthesis different snRNPs recognize sequence elements within introns, first the 5’ splice site (5‘ SS) at the intron start, then the branchpoint and at the end the 3’ splice site (3‘ SS). Multiple conformational changes and concerted assembly steps lead to formation of the active spliceosome, cleavage of the exon-intron junction, intron lariat formation and finally exon-exon ligation with cleavage of the 3’ intron-exon junction. Estimates on pre-mRNA splicing duration range from 15 sec to several minutes or, in terms of distance relative to the 3‘ SS, the earliest detected splicing events were 500 nt downstream of the 3‘ SS. However, the use of indirect assays, model genes and transcription induction/blocking leave the question of when pre-mRNA splicing of endogenous transcripts occurs unanswered. In recent years, global studies concluded that the majority of introns are removed during the course of transcription. In principal, co-transcriptional splicing reduces the need for post-transcriptional processing of the pre-mRNA. This could allow for quicker transcriptional responses to stimuli and optimal coordination between the different steps. In order to gain insight into how pre-mRNA splicing might be functionally linked to transcription, I wanted to determine when co-transcriptional splicing occurs, how transcripts with multiple introns are spliced and if and how the transcription termination process is influenced by pre-mRNA splicing. I chose two yeast species, S. cerevisiae and S. pombe, to study co-transcriptional splicing. Small genomes, short genes and introns, but very different number of intron-containing genes and multi-intron genes in S. pombe, made the combination of both model organisms a promising system to study by next-generation sequencing and to learn about co-transcriptional splicing in a broad context with applicability to other species. I used nascent RNA-Seq to characterize co-transcriptional splicing in S. pombe and developed two strategies to obtain single-molecule information on co-transcriptional splicing of endogenous genes: (1) with paired-end short read sequencing, I obtained the 3’ nascent transcript ends, which reflect the position of Pol II molecules during transcription, and the splicing status of the nascent RNAs. This is detected by sequencing the exon-intron or exon-exon junctions of the transcripts. Thus, this strategy links Pol II position with intron splicing of nascent RNA. The increase in the fraction of spliced transcripts with further distance from the intron end provides valuable information on when co-transcriptional splicing occurs. (2) with Pacific Biosciences sequencing (PacBio) of full-length nascent RNA, it is possible to determine the splicing pattern of transcripts with multiple introns, e.g. sequentially with transcription or also non-sequentially. Part of transcription termination is cleavage of the nascent transcript at the polyA site. The splicing status of cleaved and non-cleaved transcripts can provide insights into links between splicing and transcription termination and can be obtained from PacBio data. I found that co-transcriptional splicing in S. pombe is similarly prevalent to other species and that most introns are removed co-transcriptionally. Co-transcriptional splicing levels are dependent on intron position, adjacent exon length, and GC-content, but not splice site sequence. A high level of co-transcriptional splicing is correlated with high gene expression. In addition, I identified low abundance circular RNAs in intron-containing, as well as intronless genes, which could be side-products of RNA transcription and splicing. The analysis of co-transcriptional splicing patterns of 88 endogenous S. cerevisiae genes showed that the majority of intron splicing occurs within 100 nt downstream of the 3‘ SS. Saturation levels vary, and confirm results of a previous study. The onset of splicing is very close to the transcribing polymerase (within 27 nt) and implies that spliceosome assembly and conformational rearrangements must be completed immediately upon synthesis of the 3‘ SS. For S. pombe genes with multiple introns, most detected transcripts were completely spliced or completely unspliced. A smaller fraction showed partial splicing with the first intron being most often not spliced. Close to the polyA site, most transcripts were spliced, however uncleaved transcripts were often completely unspliced. This suggests a beneficial influence of pre-mRNA splicing for efficient transcript termination. Overall, sequencing of nascent RNA with the two strategies developed in this work offers significant potential for the analysis of co-transcriptional splicing, transcription termination and also RNA polymerase pausing by profiling nascent 3’ ends. I could define the position of pre-mRNA splicing during the process of transcription and provide evidence for fast and efficient co-transcriptional splicing in S. cerevisiae and S. pombe, which is associated with highly expressed genes in both organisms. Differences in S. pombe co-transcriptional splicing could be linked to gene architecture features, like intron position, GC-content and exon length.

It is currently believed that the splicing of most pre-mRNAs occurs, at least in part, co-transcriptionally. In order to validate this principle in yeast and establish an experimental system for monitoring spliceosome assembly in vivo, I have employed the chromatin immunoprecipitation (ChIP) assay to study co-transcriptional splicing events. Here, I use ChIP to examine key questions with respect to the recent proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes. In my thesis, I address: 1) whether the U1 snRNP, which binds to the 5¡¦ splice site of each intron, is recruited co-transcriptionally in vivo and 2) if so, where along the length of active genes the U1 snRNP is concentrated. U1 snRNP accumulates on downstream positions of genes containing introns but not within promoter regions or along intronless genes. More specifically, accumulation correlated with the presence and position of the intron, indicating that the intron is necessary for co-transcriptional U1 snRNP recruitment and/or retention (Kotovic et al., 2003). In contrast to capping enzymes, which bind directly to Pol II (Komarnitsky et al., 2000; Schroeder et al., 2000), the U1 snRNP is poorly detected in promoter regions, except in genes harboring promoter-proximal introns. Detection of the U1 snRNP is dependent on RNA synthesis and is abolished by intron removal. Microarray data reveals that intron-containing genes are preferentially selected by ChIP with the U1 snRNP furthermore indicating recruitment specificity to introns. Because U1 snRNP levels decrease on downstream regions of intron-containing genes with long second exons, our lab is expanding the study to 3¡¦ splice site factors in hopes to address co-transcriptional splicing. In my thesis, I also focus on questions pertaining to the requirements for recruitment of the U1 snRNP to sites of transcription. To test the proposal that the cap-binding complex (CBC) promotes U1 snRNP recognition of the 5¡¦ splice site (Colot et al., 1996), I use a ?´CBC mutant strain and determine U1 snRNP accumulation by ChIP. Surprisingly, lack of the CBC has no effect on U1 snRNP recruitment. The U1 snRNP component Prp40p has been identified as playing a pivotal role in not only cross-intron bridging (Abovich and Rosbash, 1997), but also as a link between Pol II transcription and splicing factor recruitment (Morris and Greenleaf, 2000). My data shows that Prp40p recruitment mirrors that of other U1 snRNP proteins, in that it is not detected on promoter regions, suggesting that Prp40p does not constitutively bind the phosphorylated C-terminal domain (CTD) of Pol II as previously proposed. This physical link between Pol II transcription and splicing factor recruitment is further tested in Prp40p mutant strains, in which U1 snRNP is detected at normal levels. Therefore, U1 snRNP recruitment to transcription units is not dependent on Prp40p activity. My data indicates that co-transcriptional U1 snRNP recruitment is not dependent on the CBC or Prp40p and that any effects of these players on spliceosome assembly must be reflected in later spliceosome events. My data contrasts the proposed transcription factory model in which Pol II plays a central role in the recruitment of mRNA processing factors to TUs. According to my data, splicing factor recruitment acts differently than capping enzyme and 3¡¦ end processing factor recruitment; U1 snRNP does not accumulate at promoter regions of intron-containing genes or on intronless genes rather, accumulation is based on the synthesis of the intron. These experiments have lead me to propose a kinetic model with respect to the recruitment of splicing factors to active genes. In this model, U1 snRNP accumulation at the 5¡¦ splice site requires a highly dynamic web of protein-protein and protein-RNA interactions to occur, ultimately leading to the recruitment and/or stabilization of the U1 snRNP.

Cellular function and physiology are largely established through regulated gene expression. The first step in gene expression, transcription of the genomic DNA into RNA, is a process that is highly aligned at the levels of initiation, elongation and termination. In eukaryotes, protein-coding genes are exclusively transcribed by RNA polymerase II (Pol II). Upon transcription of the first 15-20 nucleotides (nt), the emerging nascent RNA 5’ end is modified with a 7-methylguanosyl cap. This is one of several RNA modifications and processing steps that take place during transcription, i.e. co-transcriptionally. For example, protein-coding sequences (exons) are often disrupted by non-coding sequences (introns) that are removed by RNA splicing. The two transesterification reactions required for RNA splicing are catalyzed through the action of a large macromolecular machine, the spliceosome. Several non-coding small nuclear RNAs (snRNAs) and proteins form functional spliceosomal subcomplexes, termed snRNPs. Sequentially with intron synthesis different snRNPs recognize sequence elements within introns, first the 5’ splice site (5‘ SS) at the intron start, then the branchpoint and at the end the 3’ splice site (3‘ SS). Multiple conformational changes and concerted assembly steps lead to formation of the active spliceosome, cleavage of the exon-intron junction, intron lariat formation and finally exon-exon ligation with cleavage of the 3’ intron-exon junction. Estimates on pre-mRNA splicing duration range from 15 sec to several minutes or, in terms of distance relative to the 3‘ SS, the earliest detected splicing events were 500 nt downstream of the 3‘ SS. However, the use of indirect assays, model genes and transcription induction/blocking leave the question of when pre-mRNA splicing of endogenous transcripts occurs unanswered. In recent years, global studies concluded that the majority of introns are removed during the course of transcription. In principal, co-transcriptional splicing reduces the need for post-transcriptional processing of the pre-mRNA. This could allow for quicker transcriptional responses to stimuli and optimal coordination between the different steps. In order to gain insight into how pre-mRNA splicing might be functionally linked to transcription, I wanted to determine when co-transcriptional splicing occurs, how transcripts with multiple introns are spliced and if and how the transcription termination process is influenced by pre-mRNA splicing. I chose two yeast species, S. cerevisiae and S. pombe, to study co-transcriptional splicing. Small genomes, short genes and introns, but very different number of intron-containing genes and multi-intron genes in S. pombe, made the combination of both model organisms a promising system to study by next-generation sequencing and to learn about co-transcriptional splicing in a broad context with applicability to other species. I used nascent RNA-Seq to characterize co-transcriptional splicing in S. pombe and developed two strategies to obtain single-molecule information on co-transcriptional splicing of endogenous genes: (1) with paired-end short read sequencing, I obtained the 3’ nascent transcript ends, which reflect the position of Pol II molecules during transcription, and the splicing status of the nascent RNAs. This is detected by sequencing the exon-intron or exon-exon junctions of the transcripts. Thus, this strategy links Pol II position with intron splicing of nascent RNA. The increase in the fraction of spliced transcripts with further distance from the intron end provides valuable information on when co-transcriptional splicing occurs. (2) with Pacific Biosciences sequencing (PacBio) of full-length nascent RNA, it is possible to determine the splicing pattern of transcripts with multiple introns, e.g. sequentially with transcription or also non-sequentially. Part of transcription termination is cleavage of the nascent transcript at the polyA site. The splicing status of cleaved and non-cleaved transcripts can provide insights into links between splicing and transcription termination and can be obtained from PacBio data. I found that co-transcriptional splicing in S. pombe is similarly prevalent to other species and that most introns are removed co-transcriptionally. Co-transcriptional splicing levels are dependent on intron position, adjacent exon length, and GC-content, but not splice site sequence. A high level of co-transcriptional splicing is correlated with high gene expression. In addition, I identified low abundance circular RNAs in intron-containing, as well as intronless genes, which could be side-products of RNA transcription and splicing. The analysis of co-transcriptional splicing patterns of 88 endogenous S. cerevisiae genes showed that the majority of intron splicing occurs within 100 nt downstream of the 3‘ SS. Saturation levels vary, and confirm results of a previous study. The onset of splicing is very close to the transcribing polymerase (within 27 nt) and implies that spliceosome assembly and conformational rearrangements must be completed immediately upon synthesis of the 3‘ SS. For S. pombe genes with multiple introns, most detected transcripts were completely spliced or completely unspliced. A smaller fraction showed partial splicing with the first intron being most often not spliced. Close to the polyA site, most transcripts were spliced, however uncleaved transcripts were often completely unspliced. This suggests a beneficial influence of pre-mRNA splicing for efficient transcript termination. Overall, sequencing of nascent RNA with the two strategies developed in this work offers significant potential for the analysis of co-transcriptional splicing, transcription termination and also RNA polymerase pausing by profiling nascent 3’ ends. I could define the position of pre-mRNA splicing during the process of transcription and provide evidence for fast and efficient co-transcriptional splicing in S. cerevisiae and S. pombe, which is associated with highly expressed genes in both organisms. Differences in S. pombe co-transcriptional splicing could be linked to gene architecture features, like intron position, GC-content and exon length.

It is currently believed that the splicing of most pre-mRNAs occurs, at least in part, co-transcriptionally. In order to validate this principle in yeast and establish an experimental system for monitoring spliceosome assembly in vivo, I have employed the chromatin immunoprecipitation (ChIP) assay to study co-transcriptional splicing events. Here, I use ChIP to examine key questions with respect to the recent proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes. In my thesis, I address: 1) whether the U1 snRNP, which binds to the 5¡¦ splice site of each intron, is recruited co-transcriptionally in vivo and 2) if so, where along the length of active genes the U1 snRNP is concentrated. U1 snRNP accumulates on downstream positions of genes containing introns but not within promoter regions or along intronless genes. More specifically, accumulation correlated with the presence and position of the intron, indicating that the intron is necessary for co-transcriptional U1 snRNP recruitment and/or retention (Kotovic et al., 2003). In contrast to capping enzymes, which bind directly to Pol II (Komarnitsky et al., 2000; Schroeder et al., 2000), the U1 snRNP is poorly detected in promoter regions, except in genes harboring promoter-proximal introns. Detection of the U1 snRNP is dependent on RNA synthesis and is abolished by intron removal. Microarray data reveals that intron-containing genes are preferentially selected by ChIP with the U1 snRNP furthermore indicating recruitment specificity to introns. Because U1 snRNP levels decrease on downstream regions of intron-containing genes with long second exons, our lab is expanding the study to 3¡¦ splice site factors in hopes to address co-transcriptional splicing. In my thesis, I also focus on questions pertaining to the requirements for recruitment of the U1 snRNP to sites of transcription. To test the proposal that the cap-binding complex (CBC) promotes U1 snRNP recognition of the 5¡¦ splice site (Colot et al., 1996), I use a ?´CBC mutant strain and determine U1 snRNP accumulation by ChIP. Surprisingly, lack of the CBC has no effect on U1 snRNP recruitment. The U1 snRNP component Prp40p has been identified as playing a pivotal role in not only cross-intron bridging (Abovich and Rosbash, 1997), but also as a link between Pol II transcription and splicing factor recruitment (Morris and Greenleaf, 2000). My data shows that Prp40p recruitment mirrors that of other U1 snRNP proteins, in that it is not detected on promoter regions, suggesting that Prp40p does not constitutively bind the phosphorylated C-terminal domain (CTD) of Pol II as previously proposed. This physical link between Pol II transcription and splicing factor recruitment is further tested in Prp40p mutant strains, in which U1 snRNP is detected at normal levels. Therefore, U1 snRNP recruitment to transcription units is not dependent on Prp40p activity. My data indicates that co-transcriptional U1 snRNP recruitment is not dependent on the CBC or Prp40p and that any effects of these players on spliceosome assembly must be reflected in later spliceosome events. My data contrasts the proposed transcription factory model in which Pol II plays a central role in the recruitment of mRNA processing factors to TUs. According to my data, splicing factor recruitment acts differently than capping enzyme and 3¡¦ end processing factor recruitment; U1 snRNP does not accumulate at promoter regions of intron-containing genes or on intronless genes rather, accumulation is based on the synthesis of the intron. These experiments have lead me to propose a kinetic model with respect to the recruitment of splicing factors to active genes. In this model, U1 snRNP accumulation at the 5¡¦ splice site requires a highly dynamic web of protein-protein and protein-RNA interactions to occur, ultimately leading to the recruitment and/or stabilization of the U1 snRNP.

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.

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.