Interaction of Eukaryotic Translation Initiation Factor 4G with the Nuclear Cap-Binding Complex Provides a Link between Nuclear and Cytoplasmic Functions of the m⁷Guanosine Cap

Abstract

In eukaryotes the majority of mRNAs have an m 7 G cap that is added cotranscriptionally and that plays an important role in many aspects of mRNA metabolism. The nuclear cap-binding complex (CBC; consisting of CBP20 and CBP80) mediates the stimulatory functions of the cap in pre-mRNA splicing, 3 end formation, and U snRNA export. As little is known about how nuclear CBC mediates the effects of the cap in higher eukaryotes, we have characterized proteins that interact with CBC in HeLa cell nuclear extracts as potential mediators of its function. Using cross-linking and coimmunoprecipitation, we show that eukaryotic translation initiation factor 4G (eIF4G), in addition to its function in the cytoplasm, is a nuclear CBC-interacting protein. We demonstrate that eIF4G interacts with CBC in vitro and that, in addition to its cytoplasmic localization, there is a significant nuclear pool of eIF4G in mammalian cells in vivo. Immunoprecipitation experiments suggest that, in contrast to the cytoplasmic pool, much of the nuclear eIF4G is not associated with eIF4E (translation cap binding protein of eIF4F) but is associated with CBC. While eIF4G stably associates with spliceosomes in vitro and shows close association with spliceosomal snRNPs and splicing factors in vivo, depletion studies show that it does not participate directly in the splicing reaction. Taken together the data indicate that nuclear eIF4G may be recruited to pre-mRNAs via its interaction with CBC and accompanies the mRNA to the cytoplasm, facilitating the switching of CBC for eIF4F. This may provide a mechanism to couple nuclear and cytoplasmic functions of the mRNA cap structure.RNAs transcribed by RNA polymerase II (pol II) are characterized by an inverted m 7 G(5Ј)ppp(5Ј)N cap. The capping of the pre-mRNA occurs cotranscriptionally (50, 52) and is achieved by recruitment of the capping enzyme to the phosphorylated C-terminal domain of the largest subunit of pol II (5,22,36). The cap contributes to many aspects of pol II transcript metabolism, including protection against 5Ј-3Ј exonucleases, facilitating efficient pre-mRNA splicing, 3Ј end formation, U snRNA and mRNA nuclear export, and translation of mRNAs (32,34).Two distinct families of cap binding proteins (CBPs) mediate the stimulatory effects of the cap structure. In the nucleus, the cap structure interacts with the nuclear cap-binding complex (CBC), a heterodimer consisting of two highly conserved polypeptides, CBP80 and CBP20 (20,23,54). CBC plays a direct role in pre-mRNA splicing, 3Ј end formation, and U snRNA export (reviewed in reference 32). In pre-mRNA splicing CBC promotes the association of U1 snRNP with the cap-proximal 5Ј splice site (31,33). In Saccharomyces cerevisiae, CBC interacts directly with yeast-specific components of the U1 snRNP (13,15). This contrasts with the mammalian system, where there is no evidence of a direct interaction between CBC and U1 snRNP (33). Although CBC is required for efficient 3Ј end cleavage of pre-mRNA, it is not required for polyadenylation per se (11)....

Conducting the initiation of protein synthesis: the role of eIF4G

“…This was later identified as eIF4F in which the initiation factor eIF4GI associates with eIF4E and eIF4A to form a heterotrimeric complex whose role is to promote attachment of the 43 S to the capped end of mRNAs. eIF4GI is a large modular polypeptidethat interacts with many different cellular and viral proteins such as (i) initiation factors eIF4E, eIF4A, eIF3 and the nuclear cap-binding protein CBP80 (Fortes et al, 2000;Korneeva et al, 2001Korneeva et al, , 2000Lamphear et al, 1995;Mader et al, 1995;McKendrick et al, 2001) ; (ii) picornaviral proteases 2A and L (Foeger et al, 2002); (iii) viral proteins NS1 and NSP3 from influenza and rotavirus, respectively (Aragon et al, 2000;Piron et al, 1998); (iv) heat shock protein hsp27 (Cuesta et al, 2000a); (v) other proteins involved in RNA metabolism such as the poly(A) binding protein PABP (Imataka et al, 1998;Le et al, 1997;Tarun and Sachs, 1996) the decapping enzyme Dcp1 (Vilela et al, 2000), and the eIF4E kinase Mnk1 (Pyronnet et al, 1999). eIF4GI possesses three domains that are roughly equivalent in size: the N-terminal part as defined by its cleavage by picornaviral proteases, the middle 'core' domain that is critical for assembly of the translation machinery and the carboxy-terminal fragment which appears to play a modular role in translation.…”

The transformation suppressor protein Pdcd4 shuttles between nucleus and cytoplasm and binds RNA

“…Although most eIFs are located in the cytoplasm, certain eIFs appear to have additional functions in the nucleus. For example, eIF4G has been identified as part of the nuclear cap binding complex (McKendrick et al, 2001). Furthermore, there is evidence that nuclear translation occurs as part of the nonsense-mediated decay mechanism, which detects nonsense codons during a pioneer round of translation (for review, see Hentze and Kuloznik, 1999;Lykke-Andersen, 2001;Maquat and Carmichael, 2001;Schell et al, 2002).…”

Synergie entre les complexes de transcription et de maturation des ARN messagers

“…La coiffe est une structure fondamentale chez les eucaryotes où elle exerce un rôle critique dans le métabolisme de leur ARNm. On la trouve en action dans le prolongement du temps de demi vie des ARNm puisqu'elle fait office de barrière aux exonucléases qui agissent dans la direction 5'->3' [4][5][6]. L'excision de séquences introniques (processus d'épissage) est également facilitée par l'intermédiaire du complexe CBP20-CBP80 (cap binding protein 20 et 80) lié à la structure coiffe qui permet le recrutement du complexe d'épissage [5].…”

“…On la trouve en action dans le prolongement du temps de demi vie des ARNm puisqu'elle fait office de barrière aux exonucléases qui agissent dans la direction 5'->3' [4][5][6]. L'excision de séquences introniques (processus d'épissage) est également facilitée par l'intermédiaire du complexe CBP20-CBP80 (cap binding protein 20 et 80) lié à la structure coiffe qui permet le recrutement du complexe d'épissage [5]. Notons que la liaison de l'hété-rodimère à la coiffe contribue également à créer un encombrement stérique important qui constitue un obstacle aux nucléases [4].…”

“…Notons que la liaison de l'hété-rodimère à la coiffe contribue également à créer un encombrement stérique important qui constitue un obstacle aux nucléases [4]. Il a été montré que ce même intermédiaire toujours lié à la coiffe participe au transport des ARN matures du noyau vers le cytoplasme pour assurer leur traduction [4][5][6]. Enfin, la structure coiffe est d'une importance cruciale pour la traduction des protéines.…”