Mechanisms of Nuclear Genome Organization and Maintenance
- Mary Dasso, PhD, Head, Section on Cell Cycle Regulation
- Vasilisa Aksenova, PhD, Contract Research Scientist
- Alexei Arnaoutov, PhD, Staff Scientist
- Maia Ouspenskaia, DVM, Biologist
- Abik Nandi, PhD, Visiting Postdoctoral Fellow
- Sanjana Sundararajan, PhD, Visiting Postdoctoral Fellow
- Reham Aljumaah, BA, Predoctoral Intramural Research Training Award Fellow
- Sierra Ball, BA, Postbaccalaureate Intramural Research Training Award Fellow
- Daniel Cai, BA, Postbaccalaureate Intramural Research Training Award Fellow
- Fatimatou Diouf, BA, Postbaccalaureate Intramural Research Training Award Fellow
- Carolyn Egekeze, BA, Postbaccalaureate Intramural Research Training Award Fellow
- Elizabeth Giordano, BA, Postbaccalaureate Intramural Research Training Award Fellow
We are interested in mechanisms of genome maintenance and organization. During interphase, chromosomes are surrounded by the nuclear envelope (NE), which separates the nucleus from the cytoplasmic compartment of the cell. The sequestration of chromosomes within the nucleus has profound consequences for almost all aspects of gene expression and cell function. Communication between the nucleus and cytoplasm occurs through conduits called nuclear pore complexes (NPCs), which are embedded in the NE and consist of about 34 proteins called nucleoporins. Beyond nucleo-cytoplasmic trafficking, nucleoporins are important for chromosome organization, transcriptional control, RNA processing, cell signaling, and cell-cycle control. Both nucleoporins and soluble components of the nuclear trafficking machinery also perform transport-independent functions in mitotic chromosome segregation. The involvement of nucleoporins in such diverse events offers the intriguing possibility that they might coordinate these processes with nuclear trafficking and with each other. Moreover, nucleoporin dysfunction has important clinical implications: nucleoporin genes are frequently mis-regulated in cancers, and nucleoporin mutations cause congenital defects, pediatric nephrotic syndromes, and premature ovarian insufficiency. Nucleoporins are critical viral targets, and their disruption contributes to neurodegenerative conditions, including amyotrophic lateral sclerosis, frontotemporal dementia, and Huntington’s disease. Our goal is to define the biochemical roles of individual nucleoporins and understand how their dysregulation causes human disease.
Selective degradation of nucleo-cytoplasmic transport proteins
Understanding the activities of individual nucleoporins has been complicated by their multifaceted nature, abundance, and unusual stability. To overcome these issues, we employed strategies for selective and rapid degradation of individual proteins within human tissue-culture cells. Specifically, we used CRISPR-Cas9 to construct cell lines in which sequences encoding auxin-induced degron (AID) domains are inserted into both alleles of targeted genes within cells that also stably express the transport inhibitor response 1 (TIR1) protein. TIR1 promotes rapid, selective degradation of AID–tagged proteins upon addition of the plant hormone auxin. We were successful in developing cell lines that allow conditional depletions of nucleoporins associated with different regions of the NPC.
Our recent investigations regarding the roles of nucleoporins aim to address three issues. First, we are studying the role of individual nucleoporins in NPC assembly and stability. Our results indicate that different regions of the NPC can persist independently after disruption of other structural domains, indicating that the NPC is surprisingly modular after it is assembled. Second, we are looking at the role of individual nucleoporins in different nuclear trafficking pathways. We are particularly interested in mRNA export that depends upon the transcription and export 2 (TREX2) complex in conjunction with the nucleoporin TPR. Third, we are analyzing the roles of nucleoporins in non-transport processes, such as mitotic cell-cycle signaling and chromosome segregation. Defining the mechanism through which individual nucleoporins contribute to each of these processes will allow us to better design future experiments examining nucleoporin function in human development and disease.
The NPC is a highly modular structure.
NPCs are built from many copies of roughly 34 distinct nucleoporins. Models of the NPC depict it as a composite of several sub-domains, which have been named the outer rings, inner ring, cytoplasmic fibrils, and nuclear basket. The outer-ring domains of the NPC are formed from the Y-complex, which contains nine core nucleoporins (SEH1, SEC13, NUP37, NUP43, NUP85, NUP96, NUP107, NUP133, and NUP160), with a tenth subunit (ELYS) required for chromatin recruitment. Other nucleoporins (NUP205, NUP188, NUP155, NUP93, and NUP35) form the inner-ring structures. The distinct roles of individual nucleoporins and their functional interactions remain poorly understood. Moreover, NPCs undergo a disassembly-reassembly cycle during mitotic division, and a lack of tools for acute manipulation of individual nucleoporins has therefore precluded the study of their roles in maintaining structures within pre-existing pores without complications from disruption of NPC assembly.
We added AID tags and fluorescent moieties by homozygously targeting gene loci encoding Y-complex and inner-ring nucleoporins. Auxin addition resulted in a rapid loss of the targeted proteins in each case, without degradation of other nucleoporins. We anticipated that loss of any Y-complex member should result in complete destabilization of the outer rings. While this was true after depletion of NUP96 or NUP107, the loss of other Y-complex members surprisingly left the outer-ring lattice in place. The findings suggest that the outer-ring structure is remarkably resistant to perturbations, once it is fully assembled, and show that its members are not of equivalent importance in sustaining its stability. Furthermore, near complete loss of the outer ring in NUP96–depleted cells did not cause collapse of the rest of the NPC, as demonstrated by immunostaining, live microscopy, and mass spectrometry. The persistence of inner-ring nucleoporins indicated resilience of the NPC structure. Interestingly, depletion of the inner-ring nucleoporin NUP188 caused an NPC disassembly that was opposite to the profile after NUP96 depletion: inner-ring components were extensively displaced, while the components of the cytosolic fibrils, outer ring, and basket were largely unaffected. Also, there was a global reduction of almost all nucleoporins upon loss of NUP93. Together, our results indicate that the inner and outer rings of the NPC form distinct and independent structures, and that NUP93 serves as an NPC lynchpin essential for both of them.
After depletion of the inner ring or outer rings, we tested whether the residual structures remained functional for the import and export of a model nuclear transport substrate. Remarkably, there were only minimal changes in both nuclear import and export rates upon loss of NUP96 or NUP188. These results indicate that persistent inner-ring or outer-ring structures could still act as conduits for vectoral nuclear trafficking and that these modules can support independent and redundant trafficking routes. The persistence of functional pores lacking a subset of canonical nucleoporins suggests that terminally differentiated cells might retain substantial nuclear trafficking even with divergent NPC composition. Differentiated cells might thus customize function through altered NPC composition, potentially modulating specific trafficking pathways or aspects of NPC activity, such as gene regulation and post-translational protein modifications.
Functional analysis of TREX2 complex subunits and their individual roles in RNA export
A series of evolutionarily conserved complexes are co-transcriptionally recruited to nascent mRNAs, facilitating their processing as well as escorting them to and through the NPC, actions that are functionally linked; a failure to perform any of them during mRNA biogenesis directly impacts both upstream and downstream events. A key player in mRNA maturation is the transcription and export 2 (TREX2) complex, which plays a central role in bridging the transcription and export machinery. The GANP subunit of TREX2 localizes within the nucleus and associates with the NPC's nuclear basket, which protrudes from the nucleoplasmic face of the NPC. The TREX2 complex is highly conserved across eukaryotes; the mammalian TREX2 complex consists of GANP (S. cerevisiae homologue = Sac3), PCID2 (Thp1), ENY2 (Sus1), DSS1 (Sem1), and either CETN2 or CETN3 (Cdc31) proteins. In vertebrates, the nuclear basket comprises three nucleoporins, called NUP153, TPR, and NUP50. Our previous studies established that TPR interacts with GANP and plays a unique role in the export of TREX2–dependent mRNAs.
Our current research focuses on the roles of individual TREX2 complex subunits. We are particularly interested in the possibility that mRNAs may vary in the subset of TREX2 subunits that they utilize during processing and export. We are currently analyzing how individual TREX2 subunits contribute to mRNA transcript retention within the nucleus, thus shaping the cellular transcriptome, and how they work with nucleoporins to license RNA transcripts at the NPC for export.
Roles of nucleoporins during cell division
The process of chromosome segregation during cell division is essential to maintain genomic integrity. Defects in chromosome segregation lead to aneuploidy, a condition in which cells possess an abnormal number of chromosomes. Aneuploidy arising from mitotic divisions is a hallmark of many solid tumors, while aneuploidy arising during meiosis contributes to human pregnancy losses and genetic disorders, including Down’s syndrome. A microtubule-based structure called the spindle mediates chromosome segregation during division. The spindle attaches to condensed chromosomes via a proteinaceous structure on the chromosomes called the kinetochore, allowing the accurate distribution of chromosomes into daughter cells at anaphase. Surprisingly, many proteins that contribute to the assembly of the kinetochore bind tightly to the NPC during interphase. Conversely, as cells undergo NPC disassembly and NE breakdown prior to mitosis, a large number of nucleoporins associate with kinetochores. We have ongoing studies that use AID-tagged cells to understand two aspects of these relationships.
First, proper chromosome attachment to spindles (biorientation) and their alignment on the metaphase plate are essential for accurate segregation. The Spindle Assembly Checkpoint (SAC) monitors binding of spindle microtubules to kinetochores. When chromosomes are not properly attached, the SAC generates a signal that prevents anaphase onset. Three proteins that are essential for this signaling pathway (Mad1, Mad2, and p31) associate with NPCs during interphase through the nucleoporin TPR, an association that is widely conserved across eukaryotic species, but whose function is poorly understood. We are currently examining the biochemistry of their association, as well as its consequences for cellular function using the AID–tagging system. Specifically, we examined co-dependent relationships among Mad1, Mad2, p31, and TPR for NPC association by depleting individual proteins. We also ascertained that association among these proteins does not activate SAC signals prior to NE breakdown, arguing against previously proposed models for SAC control. We are currently testing the idea that SAC proteins play a role in controlling interphase NPC function or regulation of nuclear trafficking. Second, kinetochores form a large, crescent-shaped structure known as the fibrous corona, prior to spindle microtubule attachment; the Y-complex is strongly recruited to this structure. As previously demonstrated for other components of the fibrous corona, we found that the stability of Y-complex association to kinetochores is lost upon inhibition of Cyclin B/Cdc2, a key mitotic kinase. We are currently using AID lines to assess the role of the Y-complex in fibrous corona structure and stability.
Publications
- Sundararajan S, Park H, Kawano S, Johansson M, Lama B, Saito-Fujita T, Saitoh N, Arnaoutov A, Dasso M, Wang Z, Clarke DJ, Azuma Y. Methylated histones on mitotic chromosomes promote Topoisomerase IIα function for high-fidelity chromosome segregation. iScience 2023 26:106743.
- Bhat P, Aksenova V, Gazzara M, Rex EA, Aslam S, Haddad C, Gao S, Esparza M, Cagatay T, Batten K, El Zahed SS, Arnaoutov A, Zhong H, Shay JW, Tolbert B, Dasso M, Lynch KW, García-Sastre A, Fontoura BMA. Influenza virus mRNAs encode determinants for nuclear export via the cellular TREX-2 complex. Nat Commun 2023 14:2304.
- Boukaba A, Liu J, Ward C, Wu Q, Arnaoutov A, Liang J, Pugacheva EM, Dasso M, Lobanenkov V, Esteban M, Strunnikov AV. Ectopic expression of meiotic cohesin generates chromosome instability in cancer cell line. Proc Natl Acad Sci USA 2022 119:e2204071119.
- LaJoie D, Turkmen AM, Mackay DR, Jensen CC, Aksenova V, Niwa M, Dasso M, Ullman KS. A role for Nup153 in nuclear assembly reveals differential requirements for targeting of nuclear envelope constituents. Mol Biol Cell 2022 31:mbcE22050189.
- Duan L, Zaepfel BL, Aksenova V, Dasso M, Rothstein JD, Kalab P, Hayes LR. Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export. Cell Rep 2022 40:111106.
- Bley CJ, Nie S, Mobbs GW, Petrovic S, Gres AT, Liu X, Mukherjee S, Harvey S, Huber FM, Lin DH, Brown B, Tang AW, Rundlet EJ, Correia AR, Chen S, Regmi SG, Stevens TA, Jette CA, Dasso M, Patke A, Palazzo AF, Kossiakoff AA, Hoelz A. Architecture of the cytoplasmic face of the nuclear pore. Science 2022 376:6598.
Collaborators
- Yoshiaki Azuma, PhD, University of Kansas, Lawrence, KS
- Beatriz Fontoura, PhD, University of Texas Southwestern Medical Center, Dallas, TX
- Amnon Harel, PhD, Azrieli Faculty of Medicine, Bar-Ilan University, Safed, Israel
- Lindsey Hayes, MD, Johns Hopkins University School of Medicine, Baltimore, MD
- André Hoelz, PhD, Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA
- Jeffrey Rothstein, MD, Brain Science Institute, Johns Hopkins University School of Medicine, Baltimore, MD
- Alexander Strunnikov, PhD, Guangzhou Institutes of Biomedicine and Health, Guangzhou, China
- Katharine Ullman, PhD, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT
Contact
For more information, email dassom@mail.nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/dasso.
