The mTOR regulated RNA-binding protein LARP1 requires PABPC1 for guided mRNA interaction.

The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth, integrating multiple signalling cues and pathways. Key among the downstream activities of mTOR is the control of the protein synthesis machinery. This is achieved, in part, via the co-ordinated regulation of mRNAs that contain a terminal oligopyrimidine tract (TOP) at their 5'ends, although the mechanisms by which this occurs downstream of mTOR signalling are still unclear. We used RNA-binding protein (RBP) capture to identify changes in the protein-RNA interaction landscape following mTOR inhibition. Upon mTOR inhibition, the binding of LARP1 to a number of mRNAs, including TOP-containing mRNAs, increased. Importantly, non-TOP-containing mRNAs bound by LARP1 are in a translationally-repressed state, even under control conditions. The mRNA interactome of the LARP1-associated protein PABPC1 was found to have a high degree of overlap with that of LARP1 and our data show that PABPC1 is required for the association of LARP1 with its specific mRNA targets. Finally, we demonstrate that mRNAs, including those encoding proteins critical for cell growth and survival, are translationally repressed when bound by both LARP1 and PABPC1

Etude du complexe CBC et ses différents rôles dans la biogenèse des ARN

Le CBC intervient dans de nombreuses étapes du métabolisme des ARN, telle que l’épissage, la maturation de l’extrémité 3’, la dégradation, l’export et la traduction. Ainsi, le CBC constitue un complexe majeur qui peut orchestrer les différentes étapes de maturation des ARN. Récemment, nous avons identifié le complexe CBCAP, composé de CBC, ARS2 et PHAX. Nous avons montré que la protéine ARS2 stimule la formation des extrémités 3’ de plusieurs familles d’ARN dont les snARN. De plus, ARS2 stimule le recrutement de PHAX sur le CBC. Ainsi, nous proposons un modèle où CBC-ARS2 stimule la formation de l’extrémité 3’ des pré-snARN et recrute PHAX pour favoriser leur export. Une autre étude a identifié un autre complexe le CBCN, constitué de CBC, Ars2, et de ZC3H18-NEXT au lieu de PHAX. CBCN recrute l’exosome et stimule la dégradation de certains ARN, comme les PROMPTS et les transcrits «read-through » des snARN et des ARNm d’histone. Ainsi, PHAX et ZC3H18 destinent leur ARN cibles vers l’export ou la dégradation. Il a été montré que PHAX reconnait et lie spécialement les ARN de petite taille. D’une manière remarquable, nos données de CLIP-Seq et de RIP suivie par des analyses avec des puces « All genes » montrent que PHAX lie aussi d’autres familles d’ARN. En effet, PHAX lie les ARNm ainsi que des ARN non-codant avec une légère préférence pour les snARN (en comparaison avec ZC3H18). Afin de mieux comprendre le rôle de PHAX et ZC3H18, j’ai tout d’abord démontré si les deux protéines se lient simultanément au CBC. Pour ce faire, J’ai réalisé des tests de compétitions entre PHAX et ZC3H18, in vivo, et j’ai montré que la surexpression de ZC3H18 déplace PHAX du CBC et vice versa. Puis en utilisant la technique de « Tethering Assays » j’ai pu montrer que PHAX et ZC3H18 ont des effets opposés sur la biogénèse des ARNm. De plus PHAX semble avoir un effet positif sur la maturation des ARNm et ce, en empêchant ZC3H18 et l’exosome d’être recruter. Nous avons aussi montré que la déplétion de PHAX et ZC3H18 a des conséquences fonctionnelles sur le taux des formes matures des snARN. Dans le but de caractériser la protéine ZC3H18, j’ai réalisé un crible double-hybride et j’ai montré que ZC3H18 interagit avec plusieurs facteurs d’épissage. J’ai aussi identifié les domaines de ZC3H18 impliqués dans ses différentes interactions. D’une manière intéressante, l’interaction de ZC3H18 avec certains facteurs d’épissage peut être exclusive à son interaction avec NEXT. De plus, des expériences de protéomique réalisés sur un des facteurs d’épissage trouvé dans le crible, montrent qu’il co-purifie au sein d’un complexe qui pourrait faire le lien entre la coiffe et la machinerie d’épissage. En accord avec ces résultats, nos données de RNA-seq montrent que la déplétion de ZC3H18 engendre un défaut d’épissage pour des introns qui sont proches de la coiffe et ceci pour un nombre restreint de gènes. Ainsi, notre travail décode davantage le rôle de la coiffe dans les différentes étapes de maturation des ARN et suggère un modèle où la séquence des transcrits naissant stimule la formation d’un complexe spécifique à cet ARN parmi plusieurs autres.The cap binding complex (CBC) plays a key role in a number of gene expression pathways and has been proposed to participate in the discrimination of RNA families. It also enhances many RNA processing steps, including transcription, splicing, 3’end formation, degradation, export and translation.Recently, we identified the CBCAP complex, composed of CBC, Ars2 and PHAX. We showed that Ars2 stimulates snRNA 3'-end processing as well as PHAX binding to the CBC, hence coupling snRNA maturation with their export. Other studies showed that the CBC and ARS2 can form another complex that contains ZC3H18-NEXT instead of PHAX. This complex, named CBCN, is a cofactor of the RNA exosome and is involved in the degradation of cryptic RNAs such as PROMPTs and read-through transcripts at histone and snRNA genes. Thus, PHAX and ZC3H18 target specific families of capped RNA toward either export or degradation. Previous studies proposed that PHAX binds specifically to small RNAs and discriminates them over other RNA species. Surprisingly, our CLIP-Seq and RIP-microarrays data showed that in contrast to expectations, PHAX was not specific for snRNAs. It also binds mRNAs as well as other non-coding RNAs and has a weak preference for snRNAs comparing to ZC3H18. To better understand the role of PHAX and ZC3H18, Ifirst determined whether PHAX and ZC3H18 can bind simultaneously to the CBC. Competitive LUMIER IPs indicated that binding of these proteins is mutually exclusive. I then used tethering assays and could show that PHAX and ZC3H18 have opposite effect on mRNA biogenesis. These data go against a model where binding of PHAX or ZC3H18 discriminate RNA families, and instead suggest promiscuous binding for these proteins. In addition, PHAX may exert a positive effect on mRNA processing by preventing binding of ZC3H18 and recruitment of the RNA exosome. Last but not least, our RT-QPCR data show that PHAX and ZC3H18 depletions have functional consequences on the level of mature snRNA, and this is due to a competition between both proteins which occur on those snRNA read-through transcripts.To further explore the role of ZC3H18, I performed a two-hybrid screen and identified several splicing factors. I could validate these interactions, identify the domains involved and show that binding of some of these factors is exclusive with that of NEXT. Importantly, proteomic experiments with one of these factors identified a complex that makes the link between the cap and the splicing machinery. In agreement, RNA-Seq analysis of ZC3H18 knock-down cells showed alterations in splicing of cap-proximal introns, for a small set of genes.Altogether, this work reveals how the multiple roles of the RNA cap are achieved at the biochemical level, and suggests that the nascent RNA sequence triggers formation of one among several mutually exclusive complexes

The splicing factor SRSF3 is functionally connected to the nuclear RNA exosome for intronless mRNA decay

Abstract The RNA exosome fulfills important functions in the processing and degradation of numerous RNAs species. However, the mechanisms of recruitment to its various nuclear substrates are poorly understood. Using Epstein-Barr virus mRNAs as a model, we have discovered a novel function for the splicing factor SRSF3 in the quality control of nuclear mRNAs. We have found that viral mRNAs generated from intronless genes are particularly unstable due to their degradation by the nuclear RNA exosome. This effect is counteracted by the viral RNA-binding protein EB2 which stabilizes these mRNAs in the nucleus and stimulates both their export to the cytoplasm and their translation. In the absence of EB2, SRSF3 participates in the destabilization of these viral RNAs by interacting with both the RNA exosome and its adaptor complex NEXT. Taken together, our results provide direct evidence for a connection between the splicing machinery and mRNA decay mediated by the RNA exosome. Our results suggest that SRSF3 aids the nuclear RNA exosome and the NEXT complex in the recognition and degradation of certain mRNAs

CBC-ARS2 stimulates 3'-end maturation of multiple RNA families and favors cap-proximal processing

International audienc

CBC–ARS2 stimulates 3′-end maturation of multiple RNA families and favors cap-proximal processing

International audienc