Eugene Valkov

Structural Basis for Binding the Trex2 Complex to Nuclear Pores, Gal1 Localisation and Mrna Export.

The conserved Sac3:Thp1:Sem1:Sus1:Cdc31 (TREX2) complex binds to nuclear pore complexes (NPCs) and, in addition to integrating mRNA nuclear export with preceding steps in the gene expression pathway, facilitates re-positioning of highly regulated actively transcribing genes (such as GAL1) to NPCs. Although TREX2 is thought to bind NPC protein Nup1, defining the precise role of this interaction has been frustrated by the complex pleiotropic phenotype exhibited by nup1Δ strains. To provide a structural framework for understanding the binding of TREX2 to NPCs and its function in the gene expression pathway, we have determined the structure of the Nup1:TREX2 interaction interface and used this information to engineer a Sac3 variant that impairs NPC binding while not compromising TREX2 assembly. This variant inhibited the NPC association of both de-repressed and activated GAL1 and also produced mRNA export and growth defects. These results indicate that the TREX2:Nup1 interaction facilitates the efficient nuclear export of bulk mRNA together with the re-positioning of GAL1 to NPCs that is required for transcriptional control that is mediated by removal of SUMO from repressors by NPC-bound Ulp1.

Structural basis for Pan3 binding to Pan2 and its function in mRNA recruitment and deadenylation

The conserved eukaryotic Pan2–Pan3 deadenylation complex shortens cytoplasmic mRNA 3′ polyA tails to regulate mRNA stability. Although the exonuclease activity resides in Pan2, efficient deadenylation requires Pan3. The mechanistic role of Pan3 is unclear. Here, we show that Pan3 binds RNA directly both through its pseudokinase/C‐terminal domain and via an N‐terminal zinc finger that binds polyA RNA specifically. In contrast, isolated Pan2 is unable to bind RNA. Pan3 binds to the region of Pan2 that links its N‐terminal WD40 domain to the C‐terminal part that contains the exonuclease, with a 2:1 stoichiometry. The crystal structure of the Pan2 linker region bound to a Pan3 homodimer shows how the unusual structural asymmetry of the Pan3 dimer is used to form an extensive high‐affinity interaction. This binding allows Pan3 to supply Pan2 with substrate polyA RNA, facilitating efficient mRNA deadenylation by the intact Pan2–Pan3 complex.

Structural basis for the assembly and disassembly of mRNA nuclear export complexes

Most of the individual components of the nuclear elements of the gene expression pathway have been identified and high-resolution structural information is becoming available for many of them. Information is also starting to become available on the larger complexes they form and is beginning to give clues about how the dynamics of their interactions generate function. Although the translocation of export-competent messenger ribonucleoprotein particles (mRNPs) through the nuclear pore transport channel that is mediated by interactions with nuclear pore proteins (nucleoporins) is relatively well understood, the precise molecular mechanisms underlying the assembly of export-competent mRNPs in the nucleus and their Dbp5-mediated disassembly in the cytoplasm is less well defined. Considerable information has been obtained on the structure of Dbp5 in its different nucleotide-bound states and in complex with Gle1 or Nup159/NUP214. Although the precise manner by which the Dbp5 ATPase cycle is coupled to mRNP remodelling remains to be established, current models capture many key details of this process. The formation of export-competent mRNPs in the nucleus remains an elusive component of this pathway and the precise nature of the remodelling that generates these mRNPs as well as detailed understanding of the molecular mechanisms by which this step is integrated with the transcriptional, splicing and polyadenylation machinery by the TREX and TREX-2 complexes remain obscure. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.

Structure of the Dcp2-Dcp1 mRNA-decapping complex in the activated conformation

The removal of the mRNA 5′ cap (decapping) by Dcp2 shuts down translation and commits mRNA to full degradation. Dcp2 activity is enhanced by activator proteins such as Dcp1 and Edc1. However, owing to conformational flexibility, the active conformation of Dcp2 and the mechanism of decapping activation have remained unknown. Here, we report a 1.6-Å-resolution crystal structure of the Schizosaccharomyces pombe Dcp2–Dcp1 heterodimer in an unprecedented conformation that is tied together by an intrinsically disordered peptide from Edc1. In this ternary complex, an unforeseen rotation of the Dcp2 catalytic domain allows residues from both Dcp2 and Dcp1 to cooperate in RNA binding, thus explaining decapping activation by increased substrate affinity. The architecture of the Dcp2–Dcp1–Edc1 complex provides a rationale for the conservation of a sequence motif in Edc1 that is also present in unrelated decapping activators, thus indicating that the presently described mechanism of decapping activation is evolutionarily conserved.

The Structures of eIF4E-eIF4G Complexes Reveal an Extended Interface to Regulate Translation Initiation.

Eukaryotic initiation factor 4G (eIF4G) plays a central role in translation initiation through its interactions with the cap-binding protein eIF4E. This interaction is a major drug target for repressing translation and is naturally regulated by 4E-binding proteins (4E-BPs). 4E-BPs and eIF4G compete for binding to the eIF4E dorsal surface via a shared canonical 4E-binding motif, but also contain auxiliary eIF4E-binding sequences, which were assumed to contact non-overlapping eIF4E surfaces. However, it is unknown how metazoan eIF4G auxiliary sequences bind eIF4E. Here, we describe crystal structures of human and Drosophila melanogaster eIF4E-eIF4G complexes, which unexpectedly reveal that the eIF4G auxiliary sequences bind to the lateral surface of eIF4E, using a similar mode to that of 4E-BPs. Our studies provide a molecular model of the eIF4E-eIF4G complex, shed light on the competition mechanism of 4E-BPs, and enable the rational design of selective eIF4G inhibitors to dampen dysregulated translation in disease.

The principal mRNA nuclear export factor NXF1:NXT1 forms a symmetric binding platform that facilitates export of retroviral CTE-RNA

The NXF1:NXT1 complex (also known as TAP:p15) is a general mRNA nuclear export factor that is conserved from yeast to humans. NXF1 is a modular protein constructed from four domains (RRM, LRR, NTF2-like and UBA domains). It is currently unclear how NXF1:NXT1 binds transcripts and whether there is higher organization of the NXF1 domains. We report here the 3.4 Å resolution crystal structure of the first three domains of human NXF1 together with NXT1 that has two copies of the complex in the asymmetric unit arranged to form an intimate domain-swapped dimer. In this dimer, the linkers between the NXF1 LRR and NTF2-like domains interact with NXT1, generating a 2-fold symmetric platform in which the RNA-binding RRM, LRR and NTF2-like domains are arranged on one face. In addition to bulk transcripts, NXF1:NXT1 also facilitates the export of unspliced retroviral genomic RNA from simple type-D retroviruses such as SRV-1 that contain a constitutive transport element (CTE), a cis-acting 2-fold symmetric RNA stem–loop motif. Complementary structural, biochemical and cellular techniques indicated that the formation of a symmetric RNA binding platform generated by dimerization of NXF1:NXT1 facilitates the recognition of CTE-RNA and promotes its nuclear export.

Distinct modes of recruitment of the CCR4–NOT complex by Drosophila and vertebrate Nanos

Nanos proteins repress the expression of target mRNAs by recruiting effector complexes through non‐conserved N‐terminal regions. In vertebrates, Nanos proteins interact with the NOT1 subunit of the CCR4–NOT effector complex through a NOT1 interacting motif (NIM), which is absent in Nanos orthologs from several invertebrate species. Therefore, it has remained unclear whether the Nanos repressive mechanism is conserved and whether it also involves direct interactions with the CCR4–NOT deadenylase complex in invertebrates. Here, we identify an effector domain (NED) that is necessary for the Drosophila melanogaster (Dm) Nanos to repress mRNA targets. The NED recruits the CCR4–NOT complex through multiple and redundant binding sites, including a central region that interacts with the NOT module, which comprises the C‐terminal domains of NOT1–3. The crystal structure of the NED central region bound to the NOT module reveals an unanticipated bipartite binding interface that contacts NOT1 and NOT3 and is distinct from the NIM of vertebrate Nanos. Thus, despite the absence of sequence conservation, the N‐terminal regions of Nanos proteins recruit CCR4–NOT to assemble analogous repressive complexes.

Structural Characterization of the Chaetomium thermophilum TREX-2 Complex and its Interaction with the mRNA Nuclear Export Factor Mex67:Mtr2

Summary The TREX-2 complex integrates mRNA nuclear export into the gene expression pathway and is based on a Sac3 scaffold to which Thp1, Sem1, Sus1, and Cdc31 bind. TREX-2 also binds the mRNA nuclear export factor, Mex67:Mtr2, through the Sac3 N-terminal region (Sac3N). Here, we characterize Chaetomium thermophilum TREX-2, show that the in vitro reconstituted complex has an annular structure, and define the structural basis for interactions between Sac3, Sus1, Cdc31, and Mex67:Mtr2. Crystal structures show that the binding of C. thermophilum Sac3N to the Mex67 NTF2-like domain (Mex67NTF2L) is mediated primarily through phenylalanine residues present in a series of repeating sequence motifs that resemble those seen in many nucleoporins, and Mlp1 also binds Mex67:Mtr2 using a similar motif. Deletion of Sac3N generated growth and mRNA export defects in Saccharomyces cerevisiae, and we propose TREX-2 and Mlp1 function to facilitate export by concentrating mature messenger ribonucleoparticles at the nuclear pore entrance.

Domain organization within the nuclear export factor Mex67:Mtr2 generates an extended mRNA binding surface

The Mex67:Mtr2 complex is the principal yeast nuclear export factor for bulk mRNA and also contributes to ribosomal subunit export. Mex67 is a modular protein constructed from four domains (RRM, LRR, NTF2-like and UBA) that have been thought to be joined by flexible linkers like beads on a string, with the RRM and LRR domains binding RNAs and the NTF2-like and UBA domains binding FG-nucleoporins to facilitate movement through nuclear pores. Here, we show that the NTF2-like domain from Saccharomyces cerevisiae Mex67:Mtr2 also contributes to RNA binding. Moreover, the 3.3 Å resolution crystal structure of the Mex67ΔUBA:Mtr2 complex, supplemented with small angle X-ray scattering data, indicated that the LRR domain has a defined spatial relationship to the Mex67NTF2L:Mtr2 region. Conversely, the RRM domain and especially the UBA domain are more mobile. The conformation assumed by the LRR and NTF2-like domains results in clusters of positively-charged residues on each becoming arranged to form a continuous interface for binding RNA on the opposite side of the complex to the region that interacts with FG-nucleoporins to facilitate passage through nuclear pores.

Structural basis for the molecular recognition of polyadenosine RNA by Nab2 Zn fingers.

The yeast poly(A) RNA binding protein, Nab2, facilitates poly(A) tail length regulation together with targeting transcripts to nuclear pores and their export to the cytoplasm. Nab2 binds polyadenosine RNA primarily through a tandem repeat of CCCH Zn fingers. We report here the 2.15 Å resolution crystal structure of Zn fingers 3–5 of Chaetomium thermophilum Nab2 bound to polyadenosine RNA and establish the structural basis for the molecular recognition of adenosine ribonucleotides. Zn fingers 3 and 5 each bind two adenines, whereas finger 4 binds only one. In each case, the purine ring binds in a surface groove, where it stacks against an aromatic side chain, with specificity being provided by a novel pattern of H-bonds, most commonly between purine N6 and a Zn-coordinated cysteine supplemented by H-bonds between purine N7 and backbone amides. Residues critical for adenine binding are conserved between species and provide a code that allows prediction of finger-binding stoichiometry based on their sequence. Moreover, these results indicate that, in addition to poly(A) tails, Nab2 can also recognize sequence motifs elsewhere in transcripts in which adenosines are placed at key positions, consistent with its function in mRNP organization and compaction as well as poly(A) tail length regulation.