Structural basis of m7GpppG binding to the nuclear cap-binding protein complex

The 7-methyl guanosine cap structure of RNA is essential for key aspects of RNA processing, including pre-mRNA splicing, 3' end formation, U snRNA transport, nonsense-mediated decay and translation. Two cap-binding proteins mediate these effects: cytosolic eIF-4E and nuclear cap-binding protein complex (CBC). The latter consists of a CBP20 subunit, which binds the cap, and a CBP80 subunit, which ensures high-affinity cap binding. Here we report the 2.1 A resolution structure of human CBC with the cap analog m7GpppG, as well as the structure of unliganded CBC. Comparisons between these structures indicate that the cap induces substantial conformational changes within the N-terminal loop of CBP20, enabling Tyr 20 to join Tyr 43 in pi-pi stacking interactions with the methylated guanosine base. CBP80 stabilizes the movement of the N-terminal loop of CBP20 and locks the CBC into a high affinity cap-binding state. The structure for the CBC bound to m7GpppG highlights interesting similarities and differences between CBC and eIF-4E, and provides insights into the regulatory mechanisms used by growth factors and other extracellular stimuli to influence the cap-binding state of the CBC.     

Large-scale induced fit recognition of an m(7)GpppG cap analogue by the human nuclear cap-binding complex

The heterodimeric nuclear cap-binding complex (CBC) binds to the 5' cap structure of RNAs in the nucleus and plays a central role in their diverse maturation steps. We describe the crystal structure at 2.1 A resolution of human CBC bound to an m(7)GpppG cap analogue. Comparison with the structure of uncomplexed CBC shows that cap binding induces co-operative folding around the dinucleotide of some 50 residues from the N- and C-terminal extensions to the central RNP domain of the small subunit CBP20. The cap-bound conformation of CBP20 is stabilized by an intricate network of interactions both to the ligand and within the subunit, as well as new interactions of the CBP20 N-terminal tail with the large subunit CBP80. Although the structure is very different from that of other known cap-binding proteins, such as the cytoplasmic cap-binding protein eIF4E, specificity for the methylated guanosine again is achieved by sandwiching the base between two aromatic residues, in this case two conserved tyrosines. Implications for the transfer of capped mRNAs to eIF4E, required for translation initiation, are discussed.     

Novel and essential subunits in the 300-kilodalton nuclear cap binding complex of Trypanosoma brucei

One of the unique aspects of RNA processing in trypanosomatid protozoa is the presence of a cap 4 structure (m7Gpppm2(6)AmpAmpCmpm3Um) at the 5' end of all mRNAs. The cap 4 becomes part of the mRNA through trans-splicing of a 39-nucleotide-long sequence donated by the spliced leader RNA. Although the cap 4 modifications are required for trans-splicing to occur, the underlying mechanism remains to be determined. We now describe an unconventional nuclear cap binding complex (CBC) in Trypanosoma brucei with an apparent molecular mass of 300 kDa and consisting of five protein components: the known CBC subunits CBP20 and importin-alpha and three novel proteins that are only present in organisms featuring a cap 4 structure and trans-splicing. Competitive binding studies are consistent with a specific interaction between the CBC and the cap 4 structure. Downregulation of several individual components of the T. brucei CBC by RNA interference demonstrated an essential function at an early step in trans-splicing. Thus, our studies are consistent with the CBC providing a mechanistic link between cap 4 modifications and trans-splicing.     

Inhibition of mRNA deadenylation by the nuclear cap binding complex (CBC)

Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3'-exoribonuclease. Here we have investigated how the cap binding complex (CBC) affects human PARN activity. We showed that CBC, via its 80-kDa subunit (CBP80), inhibited PARN, suggesting that CBC can regulate mRNA deadenylation. The CBC-mediated inhibition of PARN was cap-independent, and in keeping with this, the CBP80 subunit alone inhibited PARN. Our data suggested a new function for CBC, identified CBC as a potential regulator of PARN, and emphasized the importance of communication between the two extreme ends of the mRNA as a key strategy to regulate mRNA degradation. Based on our data, we have proposed a model for CBC-mediated regulation of PARN, which relies on an interaction between CBP80 and PARN. Association of CBC with PARN might have importance in the regulated recruitment of PARN to the nonsense-mediated decay pathway during the pioneer round of translation.     

NOVEL WAY OF CAPPING mRNA TRIMER AND STUDIES OF ITS INTERACTION WITH HUMAN NUCLEAR CAP-BINDING COMPLEX

Binding of mRNA 5′ cap by the nuclear cap-binding complex (CBC) is crucial for a wide variety of mRNA metabolic events. The interaction involving the CBP20 subunit of CBC is mediated by numerous hydrogen bonds and by stacking of the tyrosine sidechains with two first bases of the capped mRNA. To examine a possible role of a longer mRNA chain in the CBC-cap recognition, we have synthesized an mRNA tetramer using a novel way of capping an RNA trimer and determined its affinity for CBC by fluorescence titration.     

Novel way of capping mRNA trimer and studies of its interaction with human nuclear cap-binding complex

Binding of mRNA 5' cap by the nuclear cap-binding complex (CBC) is crucial for a wide variety of mRNA metabolic events. The interaction involving the CBP20 subunit of CBC is mediated by numerous hydrogen bonds and by stacking of the tyrosine sidechains with two first bases of the capped mRNA. To examine a possible role of a longer mRNA chain in the CBC-cap recognition, we have synthesized an mRNA tetramer using a novel way of capping an RNA trimer and determined its affinity for CBC by fluorescence titration.     

Interaction between the human nuclear cap-binding protein complex and hnRNP F.

hnRNP F was identified in a screen for proteins that interact with human CBP80 and CBP20, the components of the nuclear cap-binding complex (CBC). In vitro interaction studies showed that hnRNP F can bind to both CBP20 and CBP80 individually. hnRNP F and CBC bind independently to RNA, but hnRNP F binds preferentially to CBC-RNA complexes rather than to naked RNA. The hnRNP H protein, which is 78% identical to hnRNP F and also interacts with both CBP80 and CBP20 in vitro, does not discriminate between naked RNA and CBC-RNA complexes, showing that this effect is specific. Depletion of hnRNP F from HeLa cell nuclear extract decreases the efficiency of pre-mRNA splicing, a defect which can be partially compensated by addition of recombinant hnRNP F. Thus, hnRNP F is required for efficient pre-mRNA splicing in vitro and may participate in the effect of CBC on pre-mRNA splicing.     

Cdc42 stimulates RNA splicing via the S6 kinase and a novel S6 kinase target, the nuclear cap-binding complex

Cdc42 is a low molecular weight GTP-binding protein that plays a key regulatory role in a variety of cellular activities. The importance of the coordination of different cell functions by Cdc42 is underscored by the fact that a constitutively active Cdc42 mutant induces cellular transformation. In this study, we describe a novel function for Cdc42: its ability to stimulate pre-messenger RNA splicing. This activity is dependent on cysteine 37 in the effector loop of Cdc42 but is not dependent on cell growth. A likely candidate protein for mediating the Cdc42 effects on pre-mRNA splicing is the nuclear RNA cap-binding complex (CBC), which plays a key role in an early step of cap-dependent RNA splicing. Activation of the CBC by Cdc42 can be inhibited by rapamycin. Additionally, phosphatidylinositol 3-kinase and the Cdc42 effector, pp70 S6 kinase, stimulate the RNA cap-binding activity of the CBC. S6 kinase may directly target the CBC in vivo as it can phosphorylate the 80-kDa subunit of the CBC, CBP80, at residues that are subject to a growth factor-dependent and rapamycin-sensitive phosphorylation in vivo. Together these data suggest the involvement of a Cdc42-S6 kinase pathway in the regulation of RNA splicing, mediated by an increase in capped RNA binding by the CBC, as well as raise the possibility that the effects of Cdc42 on cell growth may be due in part to its regulation of RNA processing.     

eIF4G and CBP80 share a common origin and similar domain organization: implications for the structure and function of eIF4G

Eukaryotic translation initiation factor 4G (eIF4G) plays a critical role in protein expression, and is at the center of a complex regulatory network. Together with the cap-binding protein eIF4E, it recruits the small ribosomal subunit to the 5'-end of mRNA and promotes the assembly of a functional translation initiation complex, which scans along the mRNA to the translation start codon. Human eIF4G contains three consecutive HEAT domains, as well as long unstructured regions involved in multiple protein-protein interactions. Despite the accumulating data about the structure and function of eIF4G, the mechanisms of coordination and regulation of its interactions with other factors have remained largely unknown. Here, we present evidence that eIF4G and the large subunit of the nuclear cap-binding complex, CBP80, share a common origin and domain structure. We propose that the organization of the individual domains in eIF4G and CBP80 could also be conserved. The structure of CBP80, in complex with the nuclear cap-binding protein CBP20, is used to build a model for the mutual orientation of the domains in eIF4G and their interactions with other factors. The organization of the CBP80-CBP20 complex suggests how the activity of eIF4G in translation initiation could be regulated through a dynamic network of overlapping intra- and intermolecular interactions centered around the eIF4G HEAT domains.     

Binding studies of eukaryotic initiation factor eIF4E with novel mRNA dinucleotide cap analogues

Studies on the interaction of the murine translation initiation factor 4E with two new-synthesized cap-analogues, modified at C2' of 7-methylguanosine, have been performed by means of the fluorescence titration method. No difference in the binding affinity for eIF4E was observed compared with the "anti reversed" cap analogues, possessing the analogous modifications at C3'. Potential significance of the novel caps as research tools for examination of the nuclear cap binding complex CBC80/20 has been discussed.