Nuclear localization and transactivation of SYS-1/β-catenin is the result of serial gene duplications and subfunctionalizations

β-catenin is a multifunctional protein capable of mediating cell adhesion via E-cadherin and transactivation of target genes of the canonical Wnt signaling pathway. The nematode, C. elegans contains four paralogs of β-catenin which are highly specific in their functions. Though similar in overall structure, the four β-catenins are functionally distinct, each regulating different aspects of development. Of the four, SYS-1 is a key player in Wnt dependent asymmetric cell division (ACD). In ACD, a polarized mother will give rise to a daughter with high nuclear SYS-1 and another with low nuclear SYS-1. Despite sequence dissimilarity, SYS-1 shares a close structural resemblance with human β-catenin where it retains an unstructured amino-terminus (NTD) and 12 armadillo repeats. Using existing genome sequence data from several nematodes species, we find that the four β-catenin paralogs result from 3 sequential gene duplications and neofunctionalizations during nematode evolution. SYS-1, however, lacks an unstructured carboxyl-terminus (CTD) that is essential for human β-catenin transactivation processes. This work supports the hypothesis that SYS-1 compensated for the lack of CTD by acquiring novel transactivation domains with cryptic nuclear localization signals in the NTD and the first four armadillo repeats, as shown by transactivation assays in worms and yeast. Furthermore, SYS-1 regulatory domains are not localized to the NTD as in canonical β-catenin and instead spans the entire length of the protein. Truncating SYS-1 abolishes the classical SYS-1 nuclear asymmetry, resulting in daughter cells with symmetrical SYS-1 truncation localization. A screen for SYS-1 physical interactors followed by in vivo cell fate and SYS-1 localization analyses suggest that proper SYS-1 nuclear export is facilitated by XPO-1, while an interaction with IMB-3, an importin β-like protein, suggests import mechanisms. Interestingly, XPO-1 is especially required for lowering SYS-1 in the Wnt-unsignaled nucleus, suggesting a distinct mechanism for regulating asymmetric nuclear SYS-1. In summary, we provide insights on the mechanism of β-catenin evolution within nematodes and inform SYS-1 transactivation and nuclear transport.

Global Characterization of XRN 5′-3′ Exoribonucleases and Their Responses to Environmental Stresses in Plants

The XRN family of 5′-3′ Exoribonucleases is functionally conserved in eukaryotic organisms. However, the molecular evolution of XRN proteins in plants and their functions in plant response to environment stresses remain largely unexplored. In this study, we identified 23 XRN proteins in 6 representative plant species. Polygenetic analysis revealed that XRN2 was Arabidopsis-specific among these species, and additional branches outside the clades of XRN3 and XRN4 proteins, which we named as XRN5, were found in rice, maize, and soybean. However, XRN5 in soybean lost their entire 5′-3′ XRN Exoribonuclease domain. Protein conserved sequence analysis showed that XRN3/XRN2 contained potential bipartite nuclear-localization signals (NLS) while all the XRN4 proteins lost their second KR/RR motif of NLS, potentially leading to their cytoplasm localization. SIXRN3-2 contained one mutation in this second KR/RR motif, which may change their sub-cellular localization. The promoter cis-element analysis indicated that these XRN genes responded to multiple stresses and plant hormones diversely at transcriptional level. Finally, transcriptomic analysis suggested that OsXRN3 and ZmXRN3-1 were induced by low temperature, SIXRN4 and ZmXRN4 was inhibited by heat shock, and OsXRN5 and GmXRN5-2 were repressed by drought. However, in general, the expression patterns revealed the response diversity of XRNs to environment stimuli in different plant species. Taken together, this study characterized 23 XRNs with NLS variation that contributed to their sub-cellular localization and provided an overview of the XRNs response diversity to multiple environmental stresses, suggesting that XRNs could be used as potential gene editing candidates for precise stress-tolerant crop breeding.

Epigenetic Small Molecules Rescue Nucleocytoplasmic Transport and DNA Damage Phenotypes in C9ORF72 ALS/FTD

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease with available treatments only marginally slowing progression or improving survival. A hexanucleotide repeat expansion mutation in the C9ORF72 gene is the most commonly known genetic cause of both sporadic and familial cases of ALS and frontotemporal dementia (FTD). The C9ORF72 expansion mutation produces five dipeptide repeat proteins (DPRs), and while the mechanistic determinants of DPR-mediated neurotoxicity remain incompletely understood, evidence suggests that disruption of nucleocytoplasmic transport and increased DNA damage contributes to pathology. Therefore, characterizing these disturbances and determining the relative contribution of different DPRs is needed to facilitate the development of novel therapeutics for C9ALS/FTD. To this end, we generated a series of nucleocytoplasmic transport “biosensors”, composed of the green fluorescent protein (GFP), fused to different classes of nuclear localization signals (NLSs) and nuclear export signals (NESs). Using these biosensors in conjunction with automated microscopy, we investigated the role of the three most neurotoxic DPRs (PR, GR, and GA) on seven nuclear import and two export pathways. In addition to other DPRs, we found that PR had pronounced inhibitory effects on the classical nuclear export pathway and several nuclear import pathways. To identify compounds capable of counteracting the effects of PR on nucleocytoplasmic transport, we developed a nucleocytoplasmic transport assay and screened several commercially available compound libraries, totaling 2714 compounds. In addition to restoring nucleocytoplasmic transport efficiencies, hits from the screen also counteract the cytotoxic effects of PR. Selected hits were subsequently tested for their ability to rescue another C9ALS/FTD phenotype—persistent DNA double strand breakage. Overall, we found that DPRs disrupt multiple nucleocytoplasmic transport pathways and we identified small molecules that counteract these effects—resulting in increased viability of PR-expressing cells and decreased DNA damage markers in patient-derived motor neurons. Several HDAC inhibitors were validated as hits, supporting previous studies that show that HDAC inhibitors confer therapeutic effects in neurodegenerative models.

The nuclear ubiquitin ligase adaptor SPOP is a conserved regulator of C9orf72 dipeptide toxicity

A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation of the C9orf72 repeat produces dipeptide repeat proteins (DPRs). Previously, we showed that the DPRs PR50 and GR50 are highly toxic when expressed in Caenorhabditis elegans, and this toxicity depends on nuclear localization of the DPR. In an unbiased genome-wide RNA interference (RNAi) screen for suppressors of PR50 toxicity, we identified 12 genes that consistently suppressed either the developmental arrest and/or paralysis phenotype evoked by PR50 expression. All of these genes have vertebrate homologs, and 7 of 12 contain predicted nuclear localization signals. One of these genes was spop-1, the C. elegans homolog of SPOP, a nuclear localized E3 ubiquitin ligase adaptor only found in metazoans. SPOP is also required for GR50 toxicity and functions in a genetic pathway that includes cul-3, which is the canonical E3 ligase partner for SPOP. Genetic or pharmacological inhibition of SPOP in mammalian primary spinal cord motor neurons suppressed DPR toxicity without affecting DPR expression levels. Finally, we find that knockdown of bromodomain proteins in both C. elegans and mammalian neurons, which are known SPOP ubiquitination targets, suppresses the protective effect of SPOP inhibition. Together, these data suggest a model in which SPOP promotes the DPR-dependent ubiquitination and degradation of BRD proteins. We speculate the pharmacological manipulation of this pathway, which is currently underway for multiple cancer subtypes, could also represent an entry point for therapeutic intervention to treat C9orf72 FTD/ALS.

The mRNA decapping complex is buffered by nuclear localization

ABSTRACT mRNA decay is a key step in regulating the cellular proteome. Processing bodies (P-bodies) are thought to be sites of mRNA decay and/or storage. P-body units assemble into P-body granules under stress conditions. How this assembly is regulated, however, remains poorly understood. Here, we show, in the yeast Saccharomyces cerevisiae, that the translational repressor Scd6 and the decapping stimulator Edc3 act partially redundantly in P-body assembly by sequestering the Dcp1–Dcp2 (denoted Dcp1/2) decapping complex in the cytoplasm and preventing it from becoming imported into the nucleus by the karyopherin β protein Kap95. One of two nuclear localization signals in Dcp2 overlaps with the RNA-binding site, suggesting an additional mechanism to regulate Dcp1/2 localization. Nuclear Dcp1/2 does not drive mRNA decay and might be stored there as a readily releasable pool, indicating a dynamic equilibrium between cytoplasmic and nuclear Dcp1/2. Cytoplasmic Dcp1/2 is linked to Dhh1 via Edc3. Functional P-bodies are present at the endoplasmic reticulum where Dcp2 potentially acts to increase the local concentration of Dhh1 through interaction with Edc3 to drive phase separation and hence P-body formation.

C9orf72-derived arginine-rich poly-dipeptides impede phase modifiers

Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-β2 (Kapβ2) at 1:1 ratio. The nuclear magnetic resonances of Kapβ2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapβ2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.

TREM2 interacts with TDP-43 and mediates microglial neuroprotection against TDP-43-related neurodegeneration

Triggering receptor expressed on myeloid cell 2 (TREM2) is a surface receptor that, in the central nervous system, is exclusively expressed on microglia. TREM2 variants have been linked to increased risk for neurodegenerative diseases, but the functional effects of microglial TREM2 remain largely unknown. To this end, we investigated TAR-DNA binding protein 43 kDa (TDP-43)-related neurodegenerative disease via viral-mediated expression of human TDP-43 protein (hTDP-43) in neonatal and adult mice or inducible expression of hTDP43 with defective nuclear localization signals in transgenic mice. We found that TREM2 deficiency impaired microglia phagocytic clearance of pathological TDP-43, and enhanced neuronal damage and motor function impairments. Mass cytometry analysis revealed that hTDP-43 induced a TREM2-dependent subpopulation of microglia with high CD11c expression and higher phagocytic ability. Using mass spectrometry and surface plasmon resonance analysis, we further demonstrated an interaction between TDP-43 and TREM2, in vitro and in vivo, in hTDP-43-expressing transgenic mouse brains. We computationally identified the region within hTDP-43 that interacts with TREM2 and observed the potential interaction in ALS patient tissues. Our data reveal the novel interaction between TREM2 and TDP-43, highlighting that TDP-43 is a possible ligand for microglial TREM2 and the interaction mediates neuroprotection of microglial TREM2 in TDP-43-related neurodegeneration.

Mechanosensitivity of nucleocytoplasmic transport

Mechanical force controls fundamental cellular processes in health and disease, and increasing evidence shows that the nucleus both experiences and senses applied forces. Here we show that nuclear forces differentially control both passive and facilitated nucleocytoplasmic transport, setting the rules for the mechanosensitivity of shuttling proteins. We demonstrate that nuclear force increases permeability across nuclear pore complexes, with a dependence on molecular weight that is stronger for passive than facilitated diffusion. Due to this differential effect, force leads to the translocation into or out of the nucleus of cargoes within a given range of molecular weight and affinity for nuclear transport receptors. Further, we show that the mechanosensitivity of several transcriptional regulators can be both explained by this mechanism, and engineered exogenously by introducing appropriate nuclear localization signals. Our work sets a novel framework to understand mechanically induced signalling, with potential general applicability across signalling pathways and pathophysiological scenarios.

Subcellular Localization of Epstein–Barr Virus BLLF2 and Its Underlying Mechanisms

Epstein–Barr virus (EBV), the pathogen of several human malignancies, encodes many proteins required to be transported into the nucleus for viral DNA reproduction and nucleocapsids assembly in the lytic replication cycle. Here, fluorescence microscope, mutation analysis, interspecies heterokaryon assays, co-immunoprecipitation assay, RNA interference, and Western blot were performed to explore the nuclear import mechanism of EBV encoded BLLF2 protein. BLLF2 was shown to be a nucleocytoplasmic shuttling protein neither by a chromosomal region maintenance 1 (CRM1)- nor by a transporter associated with antigen processing (TAP)-dependent pathway. Yet, BLLF2’s two functional nuclear localization signals (NLSs), NLS1 (16KRQALETVPHPQNRGR31) and NLS2 (44RRPRPPVAKRRRFPR58), were identified, whereas the predicted NES was nonfunctional. Finally, BLLF2 was proven to transport into the nucleus via a Ran-dependent and importin β1-dependent pathway. This mechanism may contribute to a more extensive insight into the assembly and synthesis of EBV virions in the nucleus, thus affording a new direction for the treatment of viruses.

Ongoing global and regional adaptive evolution of SARS-CoV-2

Understanding the trends in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution is paramount to control the COVID-19 pandemic. We analyzed more than 300,000 high-quality genome sequences of SARS-CoV-2 variants available as of January 2021. The results show that the ongoing evolution of SARS-CoV-2 during the pandemic is characterized primarily by purifying selection, but a small set of sites appear to evolve under positive selection. The receptor-binding domain of the spike protein and the region of the nucleocapsid protein associated with nuclear localization signals (NLS) are enriched with positively selected amino acid replacements. These replacements form a strongly connected network of apparent epistatic interactions and are signatures of major partitions in the SARS-CoV-2 phylogeny. Virus diversity within each geographic region has been steadily growing for the entirety of the pandemic, but analysis of the phylogenetic distances between pairs of regions reveals four distinct periods based on global partitioning of the tree and the emergence of key mutations. The initial period of rapid diversification into region-specific phylogenies that ended in February 2020 was followed by a major extinction event and global homogenization concomitant with the spread of D614G in the spike protein, ending in March 2020. The NLS-associated variants across multiple partitions rose to global prominence in March to July, during a period of stasis in terms of interregional diversity. Finally, beginning in July 2020, multiple mutations, some of which have since been demonstrated to enable antibody evasion, began to emerge associated with ongoing regional diversification, which might be indicative of speciation.