Nucleus

The mechanical properties of the nucleus are critical for maintaining nuclear integrity and function. We previously showed that chromatin dominates short-extension mechanics whereas lamins provide long-extension strain stiffening. To distinguish the roles of lamin isoforms, micromanipulation nucleus force measurements were performed on isolated nuclei from lamin A/C (Lmna-/-) and lamin B1 (Lmnb1-/-) knockout mouse embryonic fibroblast cells. Lamin A/C knockout does not alter short-extension nuclear stiffness but is essential for strain stiffening at longer extensions. Oppositely, lamin B1 loss reduced short-extension stiffness due to facultative heterochromatin loss while long-extension strain stiffening was slightly increased. Loss of lamin A/C and B1 resulted in similar lamin-chromatin linkers effects as LBR did not change and LAP2{beta} decreased in both. A simulation model of a polymeric lamin shell with stiff lamin A/C and softer lamin B1 subunits can qualitatively recapitulate experimental measurements of lamin knockout cells. Lamin A/C knockout resulted in abnormal nuclear shape but not nuclear blebbing or rupture whereas lamin B1 knockout, similar to other perturbations that cause heterochromatin loss, resulted in increased nuclear blebbing and rupture. This work illuminates the distinct mechanical roles of lamin A/C and B1 in determining nuclear structure and integrity.

BackgroundLamins are major regulators of the spatial and functional organization of chromatin. Lamins at the nuclear periphery form the lamina that anchors heterochromatin to the nuclear envelope. A subpool of A-type lamins localizes in the nuclear interior, where they also bind to euchromatic genomic regions. A-type lamin properties and chromatin association are regulated by lamin-associated polypeptide 2alpha (LAP2). Here we systematically analyze, how LAP2 depletion affects chromatin organization, accessibility and gene expression on a genome-wide level. ResultsLAP2 depletion in mouse dermal fibroblasts positively and negatively affects chromatin accessibility and gene expression throughout the genome, which correlates with changes in chromatin association of A-type lamins and the nucleosomal remodeler proteins BRG1 and CHD4. In particular, A-type lamins bind to open chromatin regions close to BRG1 and CHD4 binding sites and deregulated genes, but do not directly accumulate on genes and BRG1 and CHD4-enriched sites. Unsupervised clustering of the datasets on LAP2-bound genomic regions confirms spreading of A-type lamins to active chromatin regions containing deregulated genes and an enrichment of chromatin remodelers on a subset of these genomic regions. ConclusionsLAP2 depletion in fibroblasts leads to a gross rearrangement of chromatin. Genome-wide chromatin reorganization is linked to spreading of A-type lamins to active chromatin regions and accompanied by a restriction of chromatin remodelers to a subset of active genomic regions. These changes correlate with changes in chromatin accessibility and gene expression throughout the genome, particularly in regions where lamin binding is gained in LAP2 knockout versus wildtype cells.

Mechanical cues influence cell behavior and fate and are frequently accompanied by changes in nuclear shape; however, how epithelial nuclei accommodate such deformations remains incompletely understood. Here, we investigated the formation and regulation of nuclear wrinkles (NWs), inward folds of the nuclear envelope, in human epithelial cells. Using quantitative confocal imaging in 2.5D spheroid cultures and controlled 2D monolayers, we found that NWs formed frequently in MCF10A cells but rarely in hTERT-RPE1 cells, indicating pronounced cell-type specificity. NW frequency increased with cell density and was tightly associated with coordinated geometric changes consistent with nuclear rounding. Disruption of F-actin organization, but not microtubules, robustly induced NW formation, and acute cell rounding triggered by trypsinization was sufficient to induce widespread wrinkling across multiple cell types. Live-cell imaging revealed that NWs are dynamic and reversible at low cell density but become stabilized under sustained confinement. NW formation occurred without detectable nuclear envelope rupture, DNA damage, or stress-associated histone phosphorylation. Quantitative analysis supports a passive geometric model in which redistribution of excess nuclear surface area accommodates nuclear shape remodeling, allowing epithelial nuclei to buffer mechanical constraints while preserving nuclear integrity.

BackgroundAcrodysostosis type 2 (ACRDYS2) is a rare autosomal dominant disease characterized by skeletal defects and cognitive deficit, with clinical symptoms observed in multiple other tissues including the skin. It is caused by mutations in a phosphodiesterase, PDE4D, a key regulator of cAMP/PKA (cyclic adenosine monophosphate / protein kinase A) signalling. Despite its well-defined genetic causes, the molecular mechanisms underlying the disease remain poorly understood, with studies based largely on engineered cellular models reaching conflicting interpretations. MethodsTo investigate how endogenous dynamics are affected by PDE4D mutations in unmanipulated cells, we studied PDE4D transcript and protein expression, activity and downstream signalling in native dermal fibroblast from ACRDYS2 patients and healthy controls. ResultsSignificant reduction in total PDE4D expression in patient cells was observed both at the transcript and protein level, with marked decreases in the long isoforms PDE4D4 and PDE4D7; a reduction in PDE4D9 mRNA was also observed. PDE4D enzymatic activity was reduced in ACRDYS2 fibroblasts, though total PDE activity was largely preserved. Reduced PDE4D expression was associated with an increase in the phosphorylated form of the cAMP-responsive transcription factor CREB and elevated PRKAR1A (PKA type 1 regulatory subunit alpha) transcript levels, suggesting altered downstream signalling. Interestingly, expression of the related phosphodiesterase family member PDE4B was increased, consistent with a compensatory response to reduced PDE4D function. ConclusionsThis is the first study demonstrating reduced PDE4D expression and isoform-specific dysregulation in native ACRDYS2 cells. Together, our results support a model in which reduction in PDE4D activity and compensatory changes in other PDE4 family members contribute to the molecular pathology of ACRDYS2, providing new insights into the molecular mechanisms underlying this disorder.

HP1 proteins are highly evolutionarily conserved chromatin-associated factors known to play essential roles in genome stability and nuclear organization. In mammals, three HP1 isoforms, HP1, HP1{beta} and HP1{gamma}, have been described, but their individual functions remain incompletely characterized. Here, we inactivated HP1 or HP1{beta} in different cell lines and quantified chromosomal breaks on metaphase spreads in the presence or absence of aphidicolin-induced replication stress. Loss of HP1, but not of HP1{beta}, led to a significant increase of chromosomal breaks on chromosome arms and within pericentromeric heterochromatin under these conditions. Mechanistically, loss of HP1 was associated with a reduction in replication fork velocity, suggesting that HP1 deficiency induces a replication stress that sensitizes specific genomic loci to replication perturbation. Consistent with this, HP1 loss was associated with a moderate but consistent increase in {gamma}H2AX and 53BP1 foci, an increased occurrence of DNA synthesis during mitosis, and enhanced recruitment of FANCD2, all recognized as hallmarks of common fragile site (CFS) expression. In addition, rescue experiments using a chromodomain mutant HP1 (V22M) unable to bind H3K9me3 indicated that HP1 protective function over these specific foci did not require its interaction with this histone mark. Altogether, these data indicate that, independently of its binding to H3K9me3, HP1 stabilizes specific genomic regions that behave as HP1-dependent fragile sites, at least in part by regulating replication fork progression, limiting mitotic DNA synthesis possibly by competing with FANCD2 for chromatin access at these regions.

THAP9 is a transposable element-derived gene which encodes a protein that is homologous to the active Drosophila P-element transposase (DmTNP). Both THAP9 and DmTNP possess a C-terminal domain (CTD) which is functionally uncharacterized. Sequence and structural analysis suggest that the THAP9-CTD has a novel fold which is only found in THAP9 homologs. To explore the evolutionary history and characteristics of this novel domain, exhaustive phylogenetic analysis (using MSA, structure prediction, MSTA-based clustering) was performed. THAP9-CTD homologs were more widely distributed throughout the animal kingdom in comparison to DmTNP-CTD homologs which were restricted to arthropods. Moreover, the THAP9-CTD homologs were more conserved, especially among mammals and birds and their average length increased in a class-specific manner. Comparison with the DmTNP-CTD homologs demonstrates that although their respective CTDs may have evolved independently, they both surprisingly share similar secondary structure elements consisting of three conserved helical regions made of hydrophobic residues that are predicted to make up a conserved core. The role of the respective CTDs were further investigated by creating truncation mutants lacking the CTD. Interestingly both THAP9 and DmTNP truncation mutants are still capable of DNA excision and integration suggesting that their respective CTDs are not essential for DNA transposition. Moreover, CTD truncation favours DNA integration in THAP9: this suggests that CTD acquisition during evolution may have led to THAP9 domestication as observed in other transposable element-derived genes like Rag1 and piggybac, which have similar terminal regulatory domains.

Kinase fusion proteins (KFPs) have emerged as an important group of immune receptors encoded by plant resistance genes. Here, we report a new type of gene pair that controls resistance of wheat to the blast fungus, Pyricularia oryzae. We cloned a fungal gene involved in avirulence of P. oryzae pathotype Eleusine on wheat and designated it PWT8. We also identified its corresponding resistance gene in wheat, and tentatively named it Rwt8. This resistance gene was located at the same locus as previously identified resistance genes Rwt3 and Rwt6. Molecular cloning revealed that Rwt3, Rwt6, and Rwt8 were the same gene consisting of an identical gene pair, one encoding an NLR and the other encoding a mixed lineage kinase-like (MLKL) protein. These two genes were closely linked in a head-to-head orientation and behaved as a single gene. This gene pair recognized three AVR genes, PWT3, PWT6, and PWT8, and was designated Rwt3.6.8. The distribution of Rwt3.6.8 in common wheat landraces suggested that the gene pair may have been a factor which the D genome provided to the genus Triticum to broaden its adaptability to various environments in the world, especially in Asia and Africa.

Arabidopsis Leucine-Rich Repeat-Malectin Receptor Kinase (LRR-MAL RK) genes have been previously implicated in the early stages of pollen-pistil interactions to support compatible pollen. One member, Receptor Kinase in Flowers 1 (RKF1), has been associated with roles in the stigma to support pollen hydration as well as pollen tube growth. To better understand the function of RKF1 in these processes, a yeast two-hybrid screen was conducted with the RKF1 cytosolic kinase domain. Two positive interactors identified from this screen were the Group VII Ethylene Response Factors (ERFVIIs), RELATED TO APETALA 2.12 (RAP2.12) and RAP2.3. Their putative roles in pollen-pistil interactions were investigated using the quintuple erfvii mutant, and novel pistil-mediated pollen tube callose deposition phenotypes were uncovered during the pollen tube growth stage. Loss of seven LRR-MAL RKs including RKF1 in the pistil was previously found to cause an unusual phenotype where shorter callose plugs were deposited in wildtype pollen tubes compared to that seen in wildtype Col-0 pistils. Contrary to this, wildtype pollen tubes growing through the quintuple erfvii mutant pistil deposited callose plugs that were more elongated than that seen in wildtype Col-0 pistils. Further analyses with the proteolysis 6 (prt6) mutant and RAP2.12 rescue constructs were consistent with these phenotypes providing support that RKF1 is a negative regulator of RAP2.12 and RAP2.3 in the pistil during pollen tube growth.

The rejection of self-incompatible (SI) Brassica pollen is mediated by three signaling branches that function in parallel in the stigma. The recognition of SI pollen by the stigma S-Receptor Kinase (SRK) results in activation of the ARM-Repeat-Containing 1 E3 ubiquitin ligase (ARC1) which mediates the degradation of compatibility factors, the FERONIA (FER) and ANJEA (ANJ) receptor kinases that induces ROS accumulation to inhibitory levels and the M Locus Protein Kinase (MLPK) which may also be connected to ROS production. Arabidopsis self-incompatibility is regulated by SRK as well, but the signaling events downstream of SRK following SI pollen perception are less well-understood. In this study, we evaluated the requirements of FER, ANJ and HERCULES RECEPTOR KINASE 1 (HERK1) for SI pollen rejection in the transgenic Arabidopsis thaliana SI-Col-0{psi} srka-1 line. The{psi} srka-1 T-DNA disrupting the expression of the endogenous{psi} SRKA gene was crossed into SI-Col-0 to prevent any potential SRK transgene silencing. T-DNA mutants for FER and ANJ/HERK1 were then crossed into the SI-Col-0{psi} srka-1 line. Using standard assays for pollen-stigma interactions, the SI phenotypes were assessed for the SI-Col-0 fer, SI-Col-0 anj-1 and SI-Col-0 anj-1 herk1-1 lines. Our results presented here indicated that FER and ANJ are not required in the stigma for Arabidopsis SI pollen rejection, further providing evidence for a divergence in the SI downstream signaling pathway in Arabidopsis.

Telomeres are nucleoprotein structures that protect chromosome ends and are maintained by the Telomerase Reverse Transcriptase (TERT) protein that uses a noncoding Telomerase RNA (TR) as a template. In monkeyflowers, Mimulus lewisii had an ancient TR gene duplication, synthesizing an evolutionarily atypical sequence heterogeneous telomere. How TERT interacts with both TR paralogs during telomere maintenance is unknown and answers can shed novel insights underlying telomere function. Using new genome assemblies we discovered TERT is rapidly evolving in lineages sharing the TR duplication. We investigated the functional consequences arising from the rapid evolution, first by using yeast three-hybrid and testing the physical binding between conspecific and heterospecific TERT-TR combinations. Results showed TERT binds both ancestral (TR1) and derived (TR2) TR paralogs in M. lewisii, but not in species without a functioning TR2. We located the region of TR binding to amino acids near the KRxR motif. We then combined next-generation sequencing with Telomeric Repeat Amplification Protocol and discovered M. lewisii had high telomerase activity. Comparative transcriptomics indicated no strong evidence of expression divergence in telomere maintenance genes for M. lewisii, suggesting rapid evolution shaped TERT protein sequence. In vivo activity of M. lewisii telomerase was investigated by analyzing F1 telomeres generated by crossing M. lewisii and M. verbenaceus, which doesnt have a functioning TR2. Results showed M. verbenaceus chromosome ends in the F1 had converted into M. lewisii telomeres, suggesting dominance of the M. lewisii telomerase. We demonstrate TERT-TR coevolution can have significant consequences on the evolution of plant telomeres. Significance statementTelomeres protect chromosome ends and are maintained by the telomerase complex. We discovered the catalytic component of the telomerase (TERT) was rapidly evolving in monkeyflowers (Mimulus) and studied the molecular consequences. In M. lewisii, TERT evolved lineage-specific amino acids to bind two sequence divergent telomerase RNA paralogs. Telomerase activity assay showed M. lewisii synthesized more telomere repeats compared to its sister species without the TR duplication, and transcriptomics indicated this was not due to a change in telomere maintenance gene expression. Genetic experiments in interspecies hybrids showed M. lewisii telomerase could convert chromosome ends in sister species into M. lewisii-like telomeres suggesting functional dominance. We show rapid evolution of the telomerase can have significant effects on telomere evolution.

Changes in intracellular calcium ion (Ca{superscript 2}) concentrations generate characteristic signatures that are decoded by specialized Ca{superscript 2}-binding proteins (CaBP). Although substantial progress has been made in understanding cytosolic calcium signaling pathways, calcium signaling within organelles, particularly chloroplasts, remains poorly understood, partly because only a few EF-hand CaBP have been identified in organelles. Here, we describe a novel EF-hand protein of 18 kDa, that was found to be associated with the chloroplast envelope and peroxisomal membrane and was therefore named OEF18 (ORGANELLAR EF-HAND PROTEIN OF 18 kDa). OEF18 has a very unusual structure, containing an N-terminal myristoylation site, followed by one EF-hand in the N-terminus facing to the cytosol, and a transmembrane domain in the C-terminus. OEF18 membrane-targeting was found to be mediated by ANKYRIN REPEAT-CONTAINING PROTEIN 2A (AKR2A) via the C-terminal transmembrane domain of OEF18. Furthermore, the EF-hand in OEF18 bound Ca{superscript 2} at a physiological concentration that led to a large protein conformational change, inducing oligomerization of the N-terminal part. We found that oef18 mutants accumulated less jasmonic acid (JA) and its bioactive conjugate JA-Ile, likely causing a defect in the insect herbivore response. Wild-type OEF18 complemented the herbivory phenotype of oef18 mutants, whereas an EF-hand point mutant lacking Ca{superscript 2}-binding capacity failed to restore the wild-type response. Furthermore, OEF18 was required for resistance to salt stress in combination with dark-induced senescence. Together, these results establish OEF18 as a previously unrecognized organellar Ca{superscript 2} sensor that couples Ca{superscript 2} perception to JA-mediated defense and abiotic stress responses in plants.

This study proposes a novel therapeutic strategy to suppress cancer growth by modulating the MDM2-p53 axis via Alternative Polyadenylation (APA). MDM2 normally promotes tumorigenesis by ubiquitinating and degrading the tumor suppressor p53. In cancer cells, preferential use of proximal polyadenylation signals (PAS) results in shortened 3'UTRs, allowing oncogenic transcripts like MDM2 to evade nuclear sequestration mediated by Inverted Alu (IRAlu) double-stranded RNA structures. We hypothesized that forcing distal PAS usage would elongate the MDM2 mRNA, promoting its nuclear retention and reducing protein translation, thereby restoring p53 activity. Using CRISPR-Cas9, we targeted and deleted the most frequent proximal PAS in the MDM2 3'UTR of A549 cells. Successful genome editing was confirmed via PCR. As expected, Western blot analysis showed a significant reduction in MDM2 expression in PAS-edited cells. However, experimental outcomes contradicted our initial hypothesis: edited cells exhibited higher viability under doxorubicin treatment compared to wild-type cells. Furthermore, despite decreased MDM2 levels, a concurrent reduction in phosphorylated p53 (p-p53) was observed. These unexpected results suggest that MDM2 3'UTR elongation may trigger a non-canonical regulatory mechanism that bypasses the traditional MDM2-p53 interaction. This study highlights the complexity of post-transcriptional regulation and suggests that APA-mediated gene modulation can induce unforeseen compensatory survival pathways in cancer cells, necessitating further investigation into the broader functional landscape of elongated 3'UTRs.

Topologically associating domains (TADs) are essential structural and functional modules of the genome that play a crucial role in regulating gene expression. In this study, we systematically investigated the conservation and evolution of TADs in five closely related crops, including maize, sorghum, coix, foxtail millet and broomcorn millet. Our results show that 74% of TAD boundaries are conserved between two inbred maize lines, B73 and Mo17, and that approximately 50% or more of TAD boundaries are conserved across different crop species. TAD number remains relatively stable in the face of changes in genome size. However, the length of TADs varies depending on genome size. Furthermore, we found that large-scale transposable element expansion leads to TAD expansion, while chromosomal inversions lead to TAD fusion and the formation of new TAD boundaries. Frequent chromatin interactions between subgenome chromosomes occur after whole-genome duplication. Moreover, we also found that crossovers are enriched at TAD boundaries in maize, indicating the importance of TADs as a fundamental unit during species evolution. Overall, our study provides insights into the conservation and evolution of TADs in crop genomes and their roles in genome organization and function.

GOLDEN2-like (GLK) transcription factors are key regulators of chloroplast differentiation and photosynthetic gene expression. The classical yellow mutation in Japanese morning glory (Ipomoea nil) produces yellowish-green leaves, whereas an unstable allele, yellow-mutable, produces green somatic sectors on a yellowish-green background. The gene responsible for these mutations was identified as InGLK, which encodes a GOLDEN2-like transcription factor. The stable yellow mutant carried a 4-bp frameshift insertion in InGLK, whereas two yellow-mutable lines carried the Tpn1-family transposon Tpn12 in intron 5. Excision of Tpn12 in germinal revertants left short footprints and restored the green leaf phenotype. Genome searches identified InGLK as the sole GLK gene in I. nil. Pigment analysis of green somatic reversion sectors and yellowish-green background areas showed that most of the measured photosynthetic pigments were significantly reduced in the yellowish-green background, whereas the chlorophyll a/b ratio was unchanged. Chloroplasts in the yellowish-green tissue retained thylakoid-like membranes and starch granule-like structures but had less distinct grana-like stacks and sparse stromal lamellae-like structures. Wild-type-like chloroplast ultrastructure was restored in germinal revertants. These findings show that loss of function of a single-copy GLK gene broadly reduces photosynthetic pigment accumulation and alters chloroplast internal membrane organization. The yellow mutants of I. nil therefore provide a genetic system for examining non-redundant GLK function.

Parthenogenesis of totipotent egg cells is rare, yet widespread, across the tree of life but mechanistic insights into factors that control parthenogenesis remain sparse. The Taraxacum officinale PARTHENOGENESIS (ToPAR) gene encodes a C2H2-zinc finger and EAR domain containing protein which is required for parthenogenesis and clonal seed production in apomictic dandelions. Ectopic expression of ToPAR can trigger egg cell division in lettuce and maternal haploid induction in foxtail millet, and ToPAR has been employed in a high-penetrance synthetic apomixis system in hybrid rice. To date a convenient model system to study ToPAR function has yet to be established nor has the capacity for ToPAR to trigger cell division in non-gametic cells been tested. Here, we demonstrate that expression of ToPAR in egg cells of Arabidopsis thaliana using the EGG-CELL 1.1 promoter (pAtEC1.1) causes a reduction in seed set and can trigger egg cell division without fertilization. We found that the pAtEC1.1:ToPAR transgene is rarely transmitted through the female lineage where it causes aberrant cell divisions. Expression of ToPAR in sexual embryos under the WUSCHEL RELATED HOMEOBOX 8 (AtWOX8) promoter alters cell patterning disrupting morphogenesis. Our results demonstrate that A. thaliana can be a powerful system to dissect the mode of action of ToPAR, and that gamete-specific co-factors are not essential for its function.

To promote disease resistance, plant nucleotide-binding, leucine-rich repeat (NLR) immune receptors often require paired co-receptors. In many cases, paired NLRs comprise one NLR to perceive effectors (the sensor) and another NLR to execute cell death (the helper). However, NLRs can also pair with sensor kinase fusion protein (KFP) receptors, but whether non-NLR components within such pairs can execute cell death, remains unclear. Here, we investigate the mechanism of an immune receptor pair comprising the wheat NLR Rwt3.6.8 NLR (R3NLR) and an MLKL protein, Rwt3.6.8 associated kinase (R3AK). Using Nicotiana benthamiana transient expression assays we confirmed that both R3NLR and R3AK are required for cell death in response to blast pathogen effectors PWT3, PWT6 or PWT8. Through mutational analysis we show the 4-helical bundle (4HB) domain of R3AK is required to execute cell death and R3AK can be made auto-active by perturbing the kinase catalytic active site. Activation of R3AK is also associated with a shift to a higher oligomeric state. Furthermore, as the NLR R3NLR is not actively involved in the execution of cell death we hypothesise that R3AK acts as a helper. A phylogenetic analysis indicates widespread distribution of this paired configuration in Poales. Together, this study establishes a novel resistance mechanism involving a non-canonical NLR/MLKL system. Significance StatementHere we investigate the mechanism of a novel plant immune receptor pair from wheat, R3NLR/R3AK. A nucleotide-binding, leucine-rich repeat (NLR) receptor and a mixed lineage kinase like (MLKL) protein are both required to mediate resistance to blast pathogen effector proteins PWT3, PWT6 and PWT8. Adopting a mutagenesis approach, we show that the MLKL protein executes cell death through its N-terminal 4-helical bundle domain, and this is associated with a shift to a higher oligomeric state. Mutations of conserved sequence motifs in the NLR support its role as a sensor, although effector interactions have not yet been observed. This study reveals a plant immune receptor pair that functions via a putative NLR sensor paired to a cell death executing MLKL protein.

Actinomycin D (Act D) is a global transcriptional inhibitor widely used in research and clinical practice; however, its effects on RNA-binding protein (RBP) dynamics remain poorly understood. Analysis of an RNA-seq dataset from Act D-treated HeLa cells revealed a compensatory stress response enriched in RNA metabolism, processing, and translation. Here, we investigated the effects of Act D on the subcellular localization of RBPs using HuR as a model mRNA stabilizing RBP. Short-term Act D treatment markedly increased cytoplasmic HuR localization in HCT116 and HeLa cells where the protein is known to be active. Analysis of known pathways regulating HuR nucleocytoplasmic translocation did not fully explain this redistribution, suggesting alternative mechanisms. To identify proteins proximal to HuR following Act D treatment, we performed TurboID labeling followed by LC-MS/MS in HCT116 cells. Several proteins involved in RNA regulation were identified. Probabilistic modeling highlighted FUS, an RBP with established roles in phase-separated granule dynamics, as a candidate proximal protein. The Act D-dependent interaction between HuR and FUS was interrogated using molecular dynamics simulations and validated with proximity ligation assays. Furthermore, increased cytoplasmic localization of RBPs following Act D treatment was accompanied by formation of granular structures that were relatively fluid and could be disrupted by hypotonic shock. Collectively, our findings demonstrate that Act D induces cytoplasmic redistribution of multiple RBPs and their sequestration into dynamic granular structures, revealing a previously unrecognized cellular response to transcriptional inhibition. Graphical AbstractAct D induced cytoplasmic re-localization of HuR along with FUS and other RBPs in dynamic, hypotonic shock-sensitive granular structures. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/745449v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@515322org.highwire.dtl.DTLVardef@128f0d5org.highwire.dtl.DTLVardef@db4302org.highwire.dtl.DTLVardef@10c4a2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Salinity stress affects rice productivity due to reduced growth and sodium ion toxicity. Previously, we identified a splice variant of RADIATION SENSITIVE23a (RAD23a) as the potential basis for variation in salt-tolerance in rice germplasm. RAD23 is a known moonlighting protein associated with protein degradation. To validate the role of RAD23a in salt stress response, we characterized gene edited mutant lines that targeted the UBL and UBA2 domains of this protein. Mutation in either domain promoted shoot growth under saline and control conditions. The mutants also differed from wildtype plants in Na and K accumulation in roots and shoots under salt stress. Transcriptome analysis of mutants versus wildtype showed differential transcript abundance of multiple inorganic phosphate (Pi) starvation related genes, including OsSPX2 and OsPHO2. As a result, mutants accumulate higher Pi compared to wildtype plants. The two allelic groups for RAD23a locus also differ in root and shoot phosphorus (P) content. Further, we show that RAD23a interacts with OsSPX2, a negative post-translational regulator of OsPHR2, the master regulator of Pi starvation response. Mutants have higher shoot growth and Pi levels under low Pi conditions, linking enhanced growth of mutants to increased Pi uptake. The UBA2 domain specific mutants have higher single grain weight and per plant grain weight than wildtype. In summary, we show that the RAD23a regulates differential growth, salt response and Pi uptake in rice in a domain-specific manner supporting the moonlighting roles of RAD23a in salt tolerance and phosphorus-dependent shoot growth.

The methyltransferase EZH2 (Enhancer of Zest Homolog 2) and the demethylase KDM6B (Lysine Demethylase 6B) have been associated with epithelial to mesenchymal transition (EMT) and poor prognosis in various cancers. These enzymes methylate and demethylate H3K27me3 and regulate distinct sets of genes controlling EMT induction, despite having opposite catalytic activities. This could be due to their recruitment or the modulation of their activity by partner proteins on specific loci. This work sought to identify proteins associated with chromatin and interacting with EZH2 or with KDM6B during EMT. To do so, co-immunoprecipitation and mass spectroscopy was used under TGF{beta} (Tumor growth factor {beta}) and TNF (Tumor necrosis factor ) treatment to induce EMT in A549 lung cancer cells. Surprisingly, numerous proteins related to focal adhesions were identified to interact with EZH2 or KDM6B. These proteins are part of a nuclear protein interaction network previously described as nucleo-adhesome. Among these proteins, TGFB1I1 (transforming growth factor induced peptide 1) and CSRP2 (cysteine and glycine rich protein 2) were further confirmed to interact with KDM6B in the nucleus and even more so during EMT. The target genes of these complexes were then sought by knocking down KDM6B, TGFB1I1 or CSRP2. Three genes (coding Integrin alpha 5, Laminin y2 and Matrix Metalloproteinase 9) were confirmed to be regulated by KDM6B, TGFB1I1 and CSRP2. These findings may have clinical relevance, as immunohistochemistry analyses performed on a cohort of lung cancer patients revealed increased nuclear localization of TGFB1I1 and CSRP2 in cells undergoing EMT.

Actin-related proteins (Arps) are evolutionarily ancient and broadly expressed, yet some Arps evolved in mammals and acquired germline-specific expression. Here, we investigate the germline-specific Arp "Actin-like 8" (ACTL8), which is also misexpressed in multiple cancers with unexplored molecular roles. We show that ACTL8 surprisingly localizes to the nucleus of spermatogonia in human testes and breast and lung cancer cells. In breast cancer cells, ACTL8 misexpression alters transcriptional programs primarily in cell migration pathways, and ACTL8 depletion increases migration rate. Mechanistically, we find that ACTL8 interacts with KDM5B, an H3K4 demethylase and transcriptional repressor, and demonstrate that this interaction contributes to changes in migration. Consistent with this, ACTL8 loss increases H3K4me3 at genes associated with migration, indicating transcriptional activation. These findings establish ACTL8 as a novel nuclear Arp that plays a role in KDM5B-mediated transcriptional repression, providing insight into its function in chromatin regulation in cancer and potentially spermatogenesis.