Canonical WNT signaling-dependent gating of MYC requires a noncanonical CTCF function at a distal binding site

Abnormal WNT signaling increases MYC expression in colon cancer cells in part via oncogenic super-enhancer-(OSE)-mediated gating of the active MYC to the nuclear pore in a poorly understood process. We show here that the principal tenet of the WNT-regulated MYC gating, facilitating nuclear export of the MYC mRNA, is regulated by a CTCF binding site (CTCFBS) within the OSE to confer growth advantage in HCT-116 cells. To achieve this, the CTCFBS directs the WNT-dependent trafficking of the OSE to the nuclear pore from intra-nucleoplasmic positions in a stepwise manner. Once the OSE reaches a peripheral position, which is triggered by a CTCFBS-mediated CCAT1 eRNA activation, its final stretch (≤0.7 μm) to the nuclear pore requires the recruitment of AHCTF1, a key nucleoporin, to the CTCFBS. Thus, a WNT/ß-catenin-AHCTF1-CTCF-eRNA circuit enables the OSE to promote pathological cell growth by coordinating the trafficking of the active MYC gene within the 3D nuclear architecture.

8 Å structure of the outer rings of the Xenopus laevis nuclear pore complex obtained by cryo-EM and AI

The nuclear pore complex (NPC), one of the largest protein complexes in eukaryotes, serves as a physical gate to regulate nucleocytoplasmic transport. Here, we determined the 8 Å resolution cryo-electron microscopic (cryo-EM) structure of the outer rings containing nuclear ring (NR) and cytoplasmic ring (CR) from the Xenopus laevis NPC, with local resolutions reaching 4.9 Å. With the aid of AlphaFold2, we managed to build a pseudoatomic model of the outer rings, including the Y complexes and flanking components. In this most comprehensive and accurate model of outer rings to date, the almost complete Y complex structure exhibits much tighter interaction in the hub region. In addition to two copies of Y complexes, each asymmetric subunit in CR contains five copies of Nup358, two copies of the Nup214 complex, two copies of Nup205 and one copy of newly identified Nup93, while that in NR contains one copy of Nup205, one copy of ELYS and one copy of Nup93. These in-depth structural features represent a great advance in understanding the assembly of NPCs.

Super-resolved 3D Tracking of Cargo Transport Through Nuclear Pore Complexes by Astigmatism Imaging

This protocol describes a two-color astigmatic imaging approach that enables direct 3D visualization of cargo transport trajectories relative to a super-resolved octagonal double-ring scaffold structure of the nuclear pore complex (NPC). Though astigmatism imaging is commonly achieved via a cylindrical lens, this protocol utilizes an adaptive optics (AO) system, which enables optimization of the astigmatism for the precision needs of the experiment as well as correction of the focal mismatch arising from chromatic aberrations in multi-color applications. With this approach, single particle spatial precision values in x, y, and z are typically 5-20 nm, and these depend on astigmatism, photon level and position in z. The method enables resolution of transport conduits through the ~60 nm diameter pore of NPCs by particle tracking on the millisecond timescale. The success of this approach is enabled by the high rigidity of fully active NPCs within the nuclear envelope of permeabilized cells. For a detailed application of this protocol, please refer to https://www.nature.com/articles/s41556-021-00815-6. The figure and table numbers in this protocol that are indicated with an “NCB” prefix (e.g., NCB Figure X) refer to the figures and table in this reference paper.

Structural characterization of human importin alpha 7 in its cargo-free form at 2.5 Å resolution

Shuttling of macromolecules between nucleus and cytoplasm is a tightly regulated process mediated through specific interactions between cargo and nuclear transport proteins. In the classical nuclear import pathway, importin alpha recognizes cargo exhibiting a nuclear localization signal, and this complex is transported through the nuclear pore complex by importin beta. Humans possess seven importin alpha isoforms that can be grouped into three subfamilies, with many cargoes displaying specificity towards these importin alpha isoforms. The cargo binding sites within importin alpha isoforms are highly conserved in sequence, suggesting that specificity potentially relies on structural differences. Structures of some importin alpha isoforms, both in cargo-bound and free states, have been previously solved. However, there are currently no known structures of cargo free importin alpha isoforms within subfamily 3 (importin alpha 5, 6, 7). Here, we present the first crystal structure of human importin alpha 7 lacking the IBB domain solved at 2.5 Å resolution. The structure reveals a typical importin alpha architecture comprised of ten armadillo repeats and is most structurally conserved with importin alpha 5. Very little difference in structure was observed between the cargo-bound and free states, implying that importin alpha 7 does not undergo conformational change when binding cargo. These structural insights provide a strong platform for further evaluation of structure–function relationships and understanding how isoform specificity within the importin alpha family plays a role in nuclear transport in health and disease.

Autophagy of the Nucleus in Health and Disease

Nucleophagy is an organelle-selective subtype of autophagy that targets nuclear material for degradation. The macroautophagic delivery of micronuclei to the vacuole, together with the nucleus-vacuole junction-dependent microautophagic degradation of nuclear material, were first observed in yeast. Nuclear pore complexes and ribosomal DNA are typically excluded during conventional macronucleophagy and micronucleophagy, indicating that degradation of nuclear cargo is tightly regulated. In mammals, similarly to other autophagy subtypes, nucleophagy is crucial for cellular differentiation and development, in addition to enabling cells to respond to various nuclear insults and cell cycle perturbations. A common denominator of all nucleophagic processes characterized in diverse organisms is the dependence on the core autophagic machinery. Here, we survey recent studies investigating the autophagic processing of nuclear components. We discuss nucleophagic events in the context of pathology, such as neurodegeneration, cancer, DNA damage, and ageing.

ZC3HC1 is a structural element of the nuclear basket effecting interlinkage of TPR polypeptides

The nuclear basket (NB), anchored to the nuclear pore complex (NPC), is commonly thought of as built solely of protein TPR polypeptides, the latter thus regarded as the NB's only scaffold-forming components. In the current study, we report ZC3HC1 as a second building element of the NB. Recently described as an NB-appended protein omnipresent in vertebrates, we now show that ZC3HC1, both in vivo and in vitro, enables in a step-wise fashion the recruitment of TPR subpopulations to the NB and their linkage to already NPC-anchored TPR polypeptides. We further demonstrate that the degron-mediated rapid elimination of ZC3HC1 results in the prompt detachment of the ZC3HC1-appended TPR polypeptides from the NB and their release back into the nucleoplasm again, underscoring the role of ZC3HC1 as a natural structural element of the NB. Finally, we show that ZC3HC1 can keep TPR polypeptides positioned even at sites remote from the NB, in line with ZC3HC1 functioning as a protein connecting TPR polypeptides.