TREX exposes the RNA binding domain of Nxf1 to enable mRNA export

The metazoan TREX complex is recruited to mRNA during nuclear RNA processing and functions in exporting mRNA to the cytoplasm. Nxf1 is an mRNA export receptor, which binds processed mRNA and transports it through the nuclear pore complex. At present, the relationship between TREX and Nxf1 is not understood. Here we show that Nxf1 uses an intramolecular interaction to inhibit its own RNA binding activity. When the TREX subunits Aly and Thoc5 make contact with Nxf1, Nxf1 is driven into an open conformation, exposing its RNA binding domain, allowing RNA binding. Moreover, the combined knockdown of Aly and Thoc5 drastically reduces the amount of Nxf1 bound to mRNA in vivo and also causes a severe mRNA export block. Together, our data indicate that TREX provides a license for mRNA export by driving Nxf1 into a conformation capable of binding mRNA

Luzp4 defines a new mRNA export pathway in cancer cells

Cancer testis antigens (CTAs) represented a poorly characterized group of proteins whose expression is normally restricted to testis but are frequently up-regulated in cancer cells. Here we show that one CTA, Luzp4, is an mRNA export adaptor. It associates with the TREX mRNA export complex subunit Uap56 and harbours a Uap56 binding motif, conserved in other mRNA export adaptors. Luzp4 binds the principal mRNA export receptor Nxf1, enhances its RNA binding activity and complements Alyref knockdown in vivo. Whilst Luzp4 is up-regulated in a range of tumours, it appears preferentially expressed in melanoma cells where it is required for growth

An essential role for Clp1 in assembly of polyadenylation complex CF IA and Pol II transcription termination

Polyadenylation is a co-transcriptional process that modifies mRNA 3′-ends in eukaryotes. In yeast, CF IA and CPF constitute the core 3′-end maturation complex. CF IA comprises Rna14p, Rna15p, Pcf11p and Clp1p. CF IA interacts with the C-terminal domain of RNA Pol II largest subunit via Pcf11p which links pre-mRNA 3′-end processing to transcription termination. Here, we analysed the role of Clp1p in 3′ processing. Clp1p binds ATP and interacts in CF IA with Pcf11p only. Depletion of Clp1p abolishes transcription termination. Moreover, we found that association of mutations in the ATP-binding domain and in the distant Pcf11p-binding region impair 3′-end processing. Strikingly, these mutations prevent not only Clp1p-Pcf11p interaction but also association of Pcf11p with Rna14p-Rna15p. ChIP experiments showed that Rna15p cross-linking to the 3′-end of a protein-coding gene is perturbed by these mutations whereas Pcf11p is only partially affected. Our study reveals an essential role of Clp1p in CF IA organization. We postulate that Clp1p transmits conformational changes to RNA Pol II through Pcf11p to couple transcription termination and 3′-end processing. These rearrangements likely rely on the correct orientation of ATP within Clp1p

Molecular dissection of mRNA poly(A) tail length control in yeast

In eukaryotic cells, newly synthesized mRNAs acquire a poly(A) tail that plays several fundamental roles in export, translation and mRNA decay. In mammals, PABPN1 controls the processivity of polyadenylation and the length of poly(A) tails during de novo synthesis. This regulation is less well-detailed in yeast. We have recently demonstrated that Nab2p is necessary and sufficient for the regulation of polyadenylation and that the Pab1p/PAN complex may act at a later stage in mRNA metabolism. Here, we show that the presence of both Pab1p and Nab2p in reconstituted pre-mRNA 3′-end processing reactions has no stimulating nor inhibitory effect on poly(A) tail regulation. Importantly, the poly(A)-binding proteins are essential to protect the mature mRNA from being subjected to a second round of processing. We have determined which domains of Nab2p are important to control polyadenylation and found that the RGG-box work in conjunction with the two last essential CCCH-type zinc finger domains. Finally, we have tried to delineate the mechanism by which Nab2p performs its regulation function during polyadenylation: it likely forms a complex with poly(A) tails different from a simple linear deposit of proteins as it has been observed with Pab1p

Co-transcriptional Loading of RNA Export Factors Shapes the Human Transcriptome

During gene expression, RNA export factors are mainly known for driving nucleo-cytoplasmic transport. While early studies suggested that the exon junction complex (EJC) provides a binding platform for them, subsequent work proposed that they are only recruited by the cap binding complex to the 5′ end of RNAs, as part of TREX. Using iCLIP, we show that the export receptor Nxf1 and two TREX subunits, Alyref and Chtop, are recruited to the whole mRNA co-transcriptionally via splicing but before 3′ end processing. Consequently, Alyref alters splicing decisions and Chtop regulates alternative polyadenylation. Alyref is recruited to the 5′ end of RNAs by CBC, and our data reveal subsequent binding to RNAs near EJCs. We demonstrate that eIF4A3 stimulates Alyref deposition not only on spliced RNAs close to EJC sites but also on single-exon transcripts. Our study reveals mechanistic insights into the co-transcriptional recruitment of mRNA export factors and how this shapes the human transcriptome

Rôle de l'hnRNP Nab2p dans la régulation de la polyadénylation des ARNm chez la levure Saccharomyces cerevisiae

Dans le noyau des cellules eucaryotes, les pré-messagers transcits par l'ARN polymérase II subissent une maturation de leur extrémité 3'. Celle-ci participe à l'arrêt de leur propre synthèse et conditionne l'export des ARNm matures vers le cytoplasme où ils seront traduits en protéines. Cette modification post-transcriptionnelle se déroule en deux étapes : un clivage endonucléolytique site-spécifique et une polymérisation de résidus adénosine 5' monophosphate initiée à partir de l'extrémité 3' nouvellement générée. Chez l'organisme modèle Saccharomyces cerevisiae, la longueur de la queue poly (A) synthétisée lors de cette seconde étape est régulée et atteint une taille de 60 nucléotides mais les mécanismes mis en jeu sont mal définis. Les travaux rapportés dans cette thèse traitent essentiellement de la protéine Nab2p, membre fondateur d'une nouvelle famille de protéines eucaryotes à motif CCCH liant le poly (A). Par des approches de biochimie et de biologie moléculaire, j'ai pu mettre en évidence que Nab2p utilise conjointement ses domaines zinc finger et RGG-box pour réguler la polyadénylation lors de réactions de maturation en 3' des ARN reconstituées in vitro. Les résultats présentés suggèrent aussi que, de par le complexe qu'elle forme avec la queue poly (A) synthétisée, Nab2p empêche l'ARNm mature d'être de nouveau substrat des complexes de maturation en inhibant tout reclivage et en bloquant l'accès de l'extrémité 3' de l'ARN à la poly (A) polymérase canonique nucléaire. Cette thèse renforce des travaux antérieurs menés in vivo et indiquent que la protéine Nab2p est un acteur majeur du contrôle de la longueur des queues poly(A) des ARNm chez la levure.In the nucleus of eukaryotic cells, newly described mRNA precursors are processed at their 3' end. This biochemical process is functionally linked to transcription termination and is essential for proper mRNP assembly and export to the cytoplasm. 3' end formation occurs as a two-steps reaction : a site-specific endonucleolytic cleavage followed by polymerization of adenosine-5'-monophosphate residues initiated at the newly created 3'end. In the yeast Saccharomyces cerevisiae, the length of the poly (A) tail synthesized during this second step is controlled to reach 60 nucleotides on average, but the mechanisms involved are poorly detailed. The work reported in this thesis mostly focuses on Nab2p, founding member of a new class of CCCH-containing poly(A) binding proteins. Using biochemical and biomolecular approaches, I have discovered that the zinc finger domains of Nabb2p work in conjunction with its RGG-box region to regulate mRNA polyadenylation in reconstituted 3'-end processing reactions in vitro. Also, the results presented here suggest that Nab2p binding to the synthesized poly(A) tail prevents the mature mRNA from being re-processed by the 3'-end processing complexes. It inhibits re-cleavage and denies access of the poly(A) tail 3' end to the canonical nuclear poly(A) polymerase. This thesis reinforces previous studies conducted in vivo and highlights the major role played by Nab2p in mRNA poly(A) tail length control in yeast.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Yeast mRNA Poly(A) Tail Length Control Can Be Reconstituted in Vitro in the Absence of Pab1p-dependent Poly(A) Nuclease Activity

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