Alwin Köhler

Exporting RNA from the nucleus to the cytoplasm

The transport of RNA molecules from the nucleus to the cytoplasm is fundamental for gene expression. The different RNA species that are produced in the nucleus are exported through the nuclear pore complexes via mobile export receptors. Small RNAs (such as tRNAs and microRNAs) follow relatively simple export routes by binding directly to export receptors. Large RNAs (such as ribosomal RNAs and mRNAs) assemble into complicated ribonucleoprotein (RNP) particles and recruit their exporters via class-specific adaptor proteins. Export of mRNAs is unique as it is extensively coupled to transcription (in yeast) and splicing (in metazoa). Understanding the mechanisms that connect RNP formation with export is a major challenge in the field.

Yeast Ataxin-7 links histone deubiquitination with gene gating and mRNA export.

Targeting of a gene to the nuclear pore complexes (NPCs), known as gene gating, can affect its transcriptional state. However, the mechanism underlying gene gating is poorly understood. Here, we have identified SAGA-associated Sgf73 (ref. 10), the yeast orthologue of human Ataxin-7 (ref. 11), as a regulator of histone H2B ubiquitin levels, a modification linked to both transcription initiation and elongation. Sgf73 is a key component of a minimal histone-deubiquitinating complex. Activation of the H2B deubiquitinating protease, Ubp8, is cooperative and requires complex formation with the amino-terminal zinc-finger-containing domain of Sgf73 and Sgf11–Sus1. Through a separate domain, Sgf73 mediates recruitment of the TREX-2 mRNA export factors Sac3 and Thp1 to SAGA and their stable interaction with Sus1–Cdc31. This latter step is crucial to target TREX-2 to the NPC. Loss of Sgf73 from SAGA abrogates gene gating of GAL1 and causes a GAL1 mRNA export defect. Thus, Sgf73 provides a molecular scaffold to integrate the regulation of H2B ubiquitin levels, tethering of a gene to the NPC and export of mRNA.

Sus1, Cdc31, and the Sac3 CID region form a conserved interaction platform that promotes nuclear pore association and mRNA export.

Summary The yeast Sac3:Cdc31:Sus1:Thp1 (TREX-2) complex facilitates the repositioning and association of actively transcribing genes with nuclear pores (NPCs)—“gene gating”—that is central to integrating transcription, processing, and mRNA nuclear export. We present here the crystal structure of Sus1 and Cdc31 bound to a central region of Sac3 (the CID domain) that is crucial for its function. Sac3CID forms a long, gently undulating α helix around which one Cdc31 and two Sus1 chains are wrapped. Sus1 has an articulated helical hairpin fold that facilitates its wrapping around Sac3. In vivo studies using engineered mutations that selectively disrupted binding of individual chains to Sac3 indicated that Sus1 and Cdc31 function synergistically to promote NPC association of TREX-2 and mRNA nuclear export. These data indicate Sac3CID provides a scaffold within TREX-2 to integrate interactions between protein complexes to facilitate the coupling of transcription and mRNA export during gene expression.

Gene Regulation by Nucleoporins and Links to Cancer

Nuclear pore complexes (NPCs) composed of approximately 30 individual nucleoporins form huge macromolecular assemblies in the nuclear envelope, through which bidirectional cargo movement between the nucleus and cytoplasm occurs. Beyond their transport function, NPCs can serve as docking sites for chromatin and thereby contribute to the organization of the overall topology of chromosomes in conjunction with other factors of the nuclear envelope. Recent studies suggest that gene-NPC interactions may promote both transcription and the definition of heterochromatin-euchromatin boundaries. Intriguingly, several nucleoporins were linked to cancer, mostly in the context of chromosomal translocations, which encode nucleoporin chimeras. An emerging concept is that tumor cells exploit specific properties of nucleoporins to deregulate transcription, chromatin boundaries, and essential transport-dependent regulatory circuits. This review outlines new mechanistic links between nucleoporin function and cancer pathogenesis.

The Nuclear Pore-Associated TREX-2 Complex Employs Mediator to Regulate Gene Expression

Summary Nuclear pore complexes (NPCs) influence gene expression besides their established function in nuclear transport. The TREX-2 complex localizes to the NPC basket and affects gene-NPC interactions, transcription, and mRNA export. How TREX-2 regulates the gene expression machinery is unknown. Here, we show that TREX-2 interacts with the Mediator complex, an essential regulator of RNA Polymerase (Pol) II. Structural and biochemical studies identify a conserved region on TREX-2, which directly binds the Mediator Med31/Med7N submodule. TREX-2 regulates assembly of Mediator with the Cdk8 kinase and is required for recruitment and site-specific phosphorylation of Pol II. Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent at specific genes. TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing. Our data provide mechanistic insight into how an NPC-associated adaptor complex accesses the core transcription machinery.

Nuclear pore basket proteins are tethered to the nuclear envelope and can regulate membrane curvature.

Summary Nuclear pore complexes (NPCs) are selective transport channels embedded in the nuclear envelope. The cylindrical NPC core forms a protein coat lining a highly curved membrane opening and has a basket-like structure appended to the nucleoplasmic side. How NPCs interact with lipids, promoting membrane bending and NPC integrity, is poorly understood. Here we show that the NPC basket proteins Nup1 and Nup60 directly induce membrane curvature by amphipathic helix insertion into the lipid bilayer. In a cell-free system, both Nup1 and Nup60 transform spherical liposomes into highly curved membrane structures. In vivo, high levels of the Nup1/Nup60 amphipathic helices cause deformation of the yeast nuclear membrane, whereas adjacent helical regions contribute to anchoring the basket to the NPC core. Basket amphipathic helices are functionally linked to distinct transmembrane nucleoporins of the NPC core, suggesting a key contribution to the membrane remodeling events that underlie NPC assembly.

Structural basis for the interaction between yeast Spt-Ada-Gcn5 acetyltransferase (SAGA) complex components Sgf11 and Sus1.

Sus1 is a central component of the yeast gene gating machinery, the process by which actively transcribing genes such as GAL1 become associated with nuclear pore complexes. Sus1 is a component of both the SAGA transcriptional co-activator complex and the TREX-2 complex that binds to nuclear pore complexes. TREX-2 contains two Sus1 chains that have an articulated helical hairpin fold, enabling them to wrap around an extended α-helix in Sac3, following a helical hydrophobic stripe. In SAGA, Sus1 binds to Sgf11 and has been proposed to provide a link between SAGA and TREX-2. We present here the crystal structure of the complex between Sus1 and the N-terminal region of Sgf11 that forms an extended α-helix around which Sus1 wraps in a manner that shares some similarities with the Sus1-Sac3 interface in TREX-2. However, the Sus1-binding site on Sgf11 is somewhat shorter than on Sac3 and is based on a narrower hydrophobic stripe. Engineered mutants that disrupt the Sgf11-Sus1 interaction in vitro confirm the importance of the hydrophobic helical stripe in molecular recognition. Helix α1 of the Sus1-articulated hairpin does not bind directly to Sgf11 and adopts a wide range of conformations within and between crystal forms, consistent with the presence of a flexible hinge and also with results from previous extensive mutagenesis studies (Klöckner, C., Schneider, M., Lutz, S., Jani, D., Kressler, D., Stewart, M., Hurt, E., and Köhler, A. (2009) J. Biol. Chem. 284, 12049–12056). A single Sus1 molecule cannot bind Sgf11 and Sac3 simultaneously and this, combined with the structure of the Sus1-Sgf11 complex, indicates that Sus1 forms separate subcomplexes within SAGA and TREX-2.

Mutational Uncoupling of the Role of Sus1 in Nuclear Pore Complex Targeting of an mRNA Export Complex and Histone H2B Deubiquitination

Sus1 is an evolutionary conserved protein that functions both in transcription and mRNA export and has been proposed to contribute to coupling these processes in yeast. Sus1 mediates its different roles as a component of both the histone H2B deubiquitinating module (Sus1-Sgf11-Ubp8-Sgf73) of the SAGA (Spt-Ada-Gcn5 acetyltransferase) transcriptional co-activator and the mRNA export complex, TREX-2 (Sus1-Sac3-Thp1-Cdc31). We have dissected the different functions of Sus1 with respect to its partitioning in transcription and export complexes using a mutational approach. Here we show that the sus1–10 (E18A, S19A, and G20A) and sus1–12 (V73A and D75A) alleles of Sus1 can be dissociated from TREX-2 while leaving its interaction with SAGA largely intact. Conversely, the binding to both TREX-2 and SAGA was impaired in the sus1–11 allele (G37A and W38A), in which two highly conserved residues were mutated. In vitro experiments demonstrated that dissociation of mutant Sus1 from its partners is caused by a reduced affinity toward the TREX-2 subunit, Sac3, and the SAGA factor, Sgf11, respectively. Consistent with the biochemical data, these sus1 mutant alleles showed differential genetic relationships with SAGA and mRNA export mutants. In vivo, all three sus1 mutants were impaired in targeting TREX-2 (i.e. Sac3) to the nuclear pore complexes and exhibited nuclear mRNA export defects. This study has implications for how Sus1, in combination with distinct interaction partners, can regulate diverse aspects of gene expression.

Monoubiquitination of Histone H2B Is Intrinsic to the Bre1 RING Domain-Rad6 Interaction and Augmented by a Second Rad6-binding Site on Bre1

Background: Ubiquitination of histone H2B regulates gene expression. Results: Bre1 RING domain recognizes nucleosome and directs Rad6 to specifically monoubiquitinate H2B Lys-123. Separate Bre1 domain augments ubiquitin transfer to nucleosome by Rad6 backside binding. Conclusion: Bre1 RING and non-RING domains cooperate in coupling substrate targeting and E2 activation. Significance: We provide detailed insights into the catalytic mechanism of histone H2B ubiquitination. Ubiquitin signaling on chromatin is linked to diverse aspects of genome regulation, including gene expression and DNA repair. The yeast RING E3 ligase Bre1 combines with the E2 Rad6 to monoubiquitinate histone H2B during transcription. Little is known about how Bre1 directs Rad6 toward transferring only a single ubiquitin to a specific lysine residue. Using a defined in vitro system, we show that the Bre1 RING domain interaction with Rad6 is minimally sufficient to monoubiquitinate nucleosomes at histone H2B Lys-123. In addition, we reveal a cluster of charged residues on the Bre1 RING domain that is critical for recognizing the nucleosome surface. Notably, a second Rad6 binding domain of Bre1 interacts with the E2 backside and potentiates ubiquitin transfer to the substrate. Taken together, our study establishes a molecular framework for how distinct RING and non-RING E3 elements cooperate to regulate E2 reactivity and substrate selection during gene expression.

The Inner Nuclear Membrane Is a Metabolically Active Territory that Generates Nuclear Lipid Droplets.

Summary The inner nuclear membrane (INM) encases the genome and is fused with the outer nuclear membrane (ONM) to form the nuclear envelope. The ONM is contiguous with the endoplasmic reticulum (ER), the main site of phospholipid synthesis. In contrast to the ER and ONM, evidence for a metabolic activity of the INM has been lacking. Here, we show that the INM is an adaptable membrane territory capable of lipid metabolism. S. cerevisiae cells target enzymes to the INM that can promote lipid storage. Lipid storage involves the synthesis of nuclear lipid droplets from the INM and is characterized by lipid exchange through Seipin-dependent membrane bridges. We identify the genetic circuit for nuclear lipid droplet synthesis and a role of these organelles in regulating this circuit by sequestration of a transcription factor. Our findings suggest a link between INM metabolism and genome regulation and have potential relevance for human lipodystrophy.