Deep sequencing of mRNAs (RNA-Seq) is now the preferred method for transcriptome-wide quantification of gene expression. Yet many mRNA isoforms, such as those eliminated by nonsense-mediated decay (NMD), are inherently unstable. Thus a significant drawback of steady-state RNA-Seq is that it provides marginal information on the flux through alternative splicing pathways. Measurement of such flux necessitates capture of newly made species prior to mRNA decay. One means to capture nascent mRNAs is affinity purifying either the exon junction complex (EJC) or activated spliceosomes. Late-stage spliceosomes deposit the EJC upstream of exon-exon junctions, where it remains associated until the first round of translation. As most mRNA decay pathways are translation-dependent, these EJC- or spliceosome-associated, pre-translational mRNAs should provide an accurate record of the initial population of alternate mRNA isoforms. Previous work has analyzed the protein composition and structure of pre- translational mRNPs in detail. While in the Moore lab, my project has focused on exploring the diversity of mRNA isoforms contained within these complexes. As expected, known NMD isoforms are more highly represented in pre-translational mRNPs than in RNA-Seq libraries. To investigate whether pre-translational mRNPs contain novel mRNA isoforms, we created a bioinformatics pipeline that identified thousands of previously unannotated splicing events. Though many can be attributed to “splicing noise”, others are evolutionarily-conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis.

La voie de signalisation mTOR intègre une variété de signaux environnementaux pour réguler la croissance et le métabolisme cellulaire. Cette voie est altérée dans 70% des cancers. Les inhibiteurs allostériques de mTOR, comme la rapamycine et ses dérivés (évérolimus et temsirolimus), sont administrés aux patients atteints de tumeurs métastatiques du sein, du rein et neuroendocrines. Cependant, leur efficacité reste modeste et une majorité de patients rechutent. L'utilisation de rapalogues fait donc face à deux problèmes cliniques majeurs : 1/l’absence de biomarqueur qui permette de stratifier les patients qui bénéficieraient le plus d'un traitement par rapalogues ; 2/ l’existence de plusieurs mécanismes de résistance décrits ou non. L’objectif de mon travail de thèse est d’identifier des nouveaux gènes cible des rapalogues utilisables comme biomarqueurs prédicteurs de l’efficacité du traitement ou comme cibles thérapeutiques pour vaincre la résistance.Nous avons identifié le gène TRIB3 comme cible des rapalogues. Sous traitement, son expression est diminuée dans un panel de lignées tumorales et des échantillons tumoraux. Nous avons démontré que cette régulation est indépendante de l’inhibition de la voie mTOR, mais médiée par le répresseur transcriptionnel GCF2. Des analyses protéomiques à haut débit ont identifié TRIB3 en tant que composant du spliceosome. De plus, nous avons démontré que la régulation négative de TRIB3 est nécessaire aux rapalogues pour modifier l'épissage des pré-ARNm. A l’inverse, la surexpression de TRIB3 supprime ces effets des rapalogues. En conclusion, ce travail de thèse ouvre plusieurs perspectives: 1 / l'utilisation potentielle de TRIB3 comme biomarqueur pour prédire ou évaluer l'efficacité du traitement par les rapalogues; 2 / de nouvelles opportunités thérapeutiques ciblant ces mécanismes indépendants de mTor ; 3/ la combinaison possible des rapalogues avec des composés ciblant l’épissage afin de surmonter la résistance. / The mTOR signaling pathway senses variety of environmental cues and integrates them to regulate cellular growth and metabolism. This pathway is altered in 70% of cancers. Allosteric inhibitors of mTOR like rapamycin and its derivatives (everolimus and temsirolimus) have become standard of care in patients with metastatic breast, kidney and neuroendocrine tumors. Unfortunately, their role is modest and most of patients will relapse. Thus, in clinic there are two major concerns related to the use of rapalogs: 1/ the absence of accurate biomarker to stratify patients who would benefit from rapalogs treatment; 2/ the existence of known and unknown mechanisms of resistance. Accordingly, the aim of my PhD project is to identify new target genes of rapalogs that could be used as biomarkers to predict treatment efficacy, or as therapeutic targets, to overcome resistance.We identified TRIB3 gene as a novel target of rapalogs. Upon treatment, its expression is down-regulated both in a panel of cancer cell lines and in cancer patient samples. We showed that this regulation is independent of the mTOR signaling inhibition, but relies on a transcriptional regulation via the co-repressor GCF2. High-throughput proteomic analyses identified TRIB3 as a component of the spliceosome. Additionally, we demonstrated that the down-regulation of TRIB3 is necessary for rapalogs to alter pre-mRNA splicing. In contrast, the, overexpression of TRIB3 abolishes these effects of rapalogs. In conclusion, this PhD work leads to the following important perspectives: 1/ the potential use of TRIB3 as a biomarker to predict or asses the efficacy of rapalogs treatment; 2/ new window of therapeutic possibilities by targeting this mTOR - independent mechanism of action; 3/ the potential combination of rapalogs with splicing targeting agents to overcome resistance.

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京都大学 / 新制・課程博士 / 博士(理学) / 甲第24082号 / 理博第4849号 / 新制||理||1694(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 柘植 知彦, 教授 森 和俊, 教授 川口 真也 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Retinoic acid receptor (RAR), a member of the steroid/thyroid hormone nuclear receptor superfamily, functions as a RA-dependent transcription activator bound to the RA response element (RARE) within the promoter or enhancer region of target genes. The transcriptional activity of RAR is modulated by a large number of coregulators including coactivators and corepressors. Acinus is a nuclear protein with three isoforms (Acinus-L, Acinus-S and Acinus-S'). Acinus-S' interacts with the A/B domain of RAR and represses RAR-regulated genes expression. Acinus (without isoform definition) has been identified as a component of nuclear speckles, the spliceosome and the exon junction complex (EJC), suggesting its localization in nuclear speckles and involvement in RNA processing. Acinus-S has been shown to localize in nuclear speckles. However, it is unclear whether the other two isoforms also localize in nuclear speckles. In addition, the role of Acinus in regulating pre-mRNA splicing is unclear. The goal of these studies was to examine the nuclear localization of Acinus-L and Acinus-S' and to determine the role of Acinus isoforms in RAR-dependent splicing. The sub-nuclear localization of Acinus-L and Acinus-S' was determined using fluorescence microscopy. Acinus-S' colocalizes with SC35 in nuclear speckles while Acinus-L localizes diffusely throughout the nucleoplasm. RA treatment has little effect on the sub-nuclear localization of Acinus-L and Acinus-S'. The domains/regions necessary for the distinct sub-nuclear localization of Acinus-L and Acinus-S' were identified. The speckled sub-nuclear localization of Acinus-S' is dependent on its C-terminal RS- and RD/E-rich region but is independent of the phosphorylation status of Ser-453 and Ser-604 within this region. The unique N-terminal SAP-motif of Acinus-L is responsible for its diffuse localization in the nucleus. Moreover, the sub-nuclear localization of Acinus isoforms is affected by each other, which is determined by the combinatorial effect of the more potent SAP motif of Acinus-L and the C-terminal RS- and RD/E-rich region in all Acinus isoforms. The C-terminal RS- and RD/E-rich region of Acinus mediates the colocalization of Acinus isoforms as well as with its interacting protein RNPS1. The role of Acinus isoforms in regulating pre-mRNA splicing was explored using in vivo splicing assays. Both Acinus-L and Acinus-S', with the activity of Acinus-L higher than that of Acinus-S', increase the splicing of a RA-responsive minigene containing a weak 5' splice site but not a RA-responsive minigene containing a strong 5' splice site. RA treatment further enhances the splicing activity of Acinus in a dose- and time-dependent manner, suggesting a RA-dependent activity in addition to a RA-independent activity of Acinus. The RA-independent effect of Acinus on the splicing of pre-mRNAs containing the weak 5' splice site occurs to varying degrees using minigene constructs containing several different promoters while the RA-dependent splicing activity of Acinus is specific for transcripts derived from the minigene driven by the RARE-containing promoter. This suggests that the ligand-dependent splicing activity of Acinus is related to the RA-activated RAR bound to the RARE. The ligand-dependent splicing activity of Acinus was further shown to be promoter-specific, depending on the ligand-dependent transcription activator. The RRM domain was identified to be necessary for the RA-dependent splicing activity of Acinus. The RA-independent splicing activity of Acinus is repressed by RNPS1. Unexpectedly, the C-terminal RS- and RD/E rich region is dispensable for the splicing activity of Acinus in regulating the minigene containing a weak 5' splice site. Importantly, measurement of the splicing of endogenous human RARâ and Bcl-x in vivo demonstrates that Acinus stimulates the use of the weaker alternative 5' splice site of these two genes in a RA-dependent manner for RARâ and in a RA-independent manner for Bcl-x. Taken together, these studies demonstrate the distinct sub-nuclear localization of Acinus-L and Acinus-S', and identified the domains that are responsible for their sub-nuclear localization, which shed light on possible distinct functions between Acinus isoforms. In addition, both Acinus-L and Acinus-S' have been shown to be splicing cofactors (with the activity of Acinus-L higher than that of Acinus-S') that facilitate constitutive splicing of pre-mRNAs containing a weak 5' splice site and regulate alternative splicing in favor of the isoform generated from the weaker alternative 5' splice site. Both Acinus-L and Acinus-S' have a RA-dependent splicing activity specific for RA-responsive genes, which suggests that Acinus functions in RAR-dependent splicing. / Biochemistry

The human proteome is exponentially derived from a limited number of genes via alternative splicing, where one gene gives rise to multiple proteins. Alternatively spliced gene products, although crucial for normal physiology, are also linked to an increasing number of pathologies. Consequently, a growing focus is currently being placed on elucidating the extrinsic cues and ensuing signaling mechanisms which direct changes in gene splicing to yield functionally distinct proteins. Of note is the dysregulation of the vascular endothelial growth factor (VEGF) receptor, Flt1 and its soluble splice variants, sFlt1_v1 and sFlt1_v2, in the pregnancy-related disorder, preeclampsia. Preeclampsia is characterized by proteinuria and hypertension and is responsible for almost 600,000 maternal and fetal yearly deaths, worldwide. Here, we examined the impact of endothelial mitogens VEGF and FGF2 (fibroblast growth factor 2), both of which are upregulated in preeclampsia, on Flt1 transcript variants in umbilical vein endothelial cells. We tested the hypothesis that VEGF modulates the expression of Flt1 variants via the signaling kinase Akt and its impact on SR proteins. VEGF was observed to induce expression of overall Flt1 mRNA, principally as variants Flt1 and sFlt1_v1. Conversely, FGF2 induced a shift in splicing toward sFlt1_v2 without significant increase in overall Flt1. Based on inhibitor studies, the VEGF and FGF2 signals were transduced via ERK, but with the involvement of different upstream components. We mapped predicted SR protein binding to Flt1 pre-mRNA and identified two candidate proteins, SRSF2 and SRSF3, that may be involved in VEGF- or FGF2-induced Flt1 pre-mRNA splicing. Examination of SRSF2 and SRSF3 relative mRNA expression levels, following inhibition of VEGF- and FGF2-activated kinases, indicates that FGF2 significantly downregulates SRSF3 mRNA levels via PKC-independent activation of ERK. Additionally, our data suggest that FGF2 may impact Flt1 and sFlt1_v1 via SR protein kinases Akt and SRPK, while conversely regulating sFlt1_v2 levels via Clk. We did not find evidence of VEGF-induced Flt1 variant splicing via SR protein kinase activation or SRSF2 and SRSF3 mRNA levels. Thus, VEGF and FGF2 signals were tranduced via related but distinct mechanisms to differentially influence Flt1 pre-mRNA splicing. These findings implicate VEGF and FGF2 and their related intracellular signaling mechanisms in soluble Flt1 regulation. / Ph. D.

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.