Selective recognition of PTRE1 transcripts mediated by protein-protein interaction between the m6A reader ECT2 and PTRE1.

N6-methyladenosine (m6A) is a prevalent internal post-transcriptional modification in eukaryotic RNAs executed by m6A-binding proteins known as "readers." Our previous research demonstrated that the Arabidopsis m6A reader ECT2 positively regulates transcript levels of the proteasome regulator PTRE1 and several 20S proteasome subunits, thereby enhancing 26S proteasome activity. However, mechanism underlying the selective recognition of m6A targets by readers, such as ECT2, remains elusive. In this study, we further demonstrate that ECT2 physically interacts with PTRE1 and several 20S proteasome subunits. This interaction, which occurs on the ribosome, involves the N terminus of PTRE1, suggesting that ECT2 might bind to the nascent PTRE1 polypeptide. Deleting ECT2's protein interaction domain impairs its mRNA-binding ability, whereas mutations in the m6A-RNA-binding site do not affect protein-protein interactions. Moreover, introducing a novel protein-binding domain into ECT2 increases transcript levels of proteins interacting with this domain. Our findings indicate that interaction with the PTRE1 protein enhances ECT2's binding to PTRE1 m6A mRNAs during translation, thereby regulating PTRE1 mRNA levels.

Elevated expression of ECT2 as a diagnostic marker and prognostic indicator in endometrial cancer.

OBJECTIVES: The study aims to investigate genes associated with endometrial cancer (EC) progression to identify new biomarkers for early detection. METHODS: Differentially expressed genes (DEGs), Series test of cluster (STC) and protein-protein interaction analyses identified hub genes in EC. Clinical samples were utilized to examine the expression pattern of ECT2, assess its prognostic value, and evaluate its diagnostic potential. RESULTS: Upregulated DEGs were significantly enriched in cancer-related processes and pathways. Validations across databases identified ASPM, ATAD2, BUB1B, ECT2, KIF14, NUF2, NCAPG, and SPAG5 as potential hub genes, with ECT2 exhibiting the highest diagnostic efficacy. The expression levels of ECT2 varied significantly across different clinical stages, pathological grades, and metastasis statuses in UCEC. Furthermore, ECT2 mRNA was upregulated in the p53abn group, indicating a poorer prognosis, and downregulated in the MMRd and NSMP groups, suggesting a moderate prognosis. In clinical samples, ECT2 expression increased from normal endometria and endometrial hyperplasia without atypia (EH) to atypical endometrial hyperplasia (AH) and EC, effectively distinguishing between benign and malignant endometria. High ECT2 expression was associated with an unfavourable prognosis. CONCLUSIONS: ECT2 expression significantly rises in AH and EC, showing high accuracy in distinguishing between benign and malignant endometria. ECT2 emerges as a promising biomarker for diagnosing endometrial neoplasia and as a prognostic indicator in EC.

1,25-Dihydroxyvitamin D3 Suppresses Prognostic Survival Biomarkers Associated with Cell Cycle and Actin Organization in a Non-Malignant African American Prostate Cell Line.

Vitamin D3 is a steroid hormone that confers anti-tumorigenic properties in prostate cells. Serum vitamin D3 deficiency has been associated with advanced prostate cancer (PCa), particularly affecting African American (AA) men. Therefore, elucidating the pleiotropic effects of vitamin D on signaling pathways, essential to maintaining non-malignancy, may provide additional drug targets to mitigate disparate outcomes for men with PCa, especially AA men. We conducted RNA sequencing on an AA non-malignant prostate cell line, RC-77N/E, comparing untreated cells to those treated with 10 nM of vitamin D3 metabolite, 1α,25(OH)2D3, at 24 h. Differential gene expression analysis revealed 1601 significant genes affected by 1α,25(OH)2D3 treatment. Pathway enrichment analysis predicted 1α,25(OH)2D3- mediated repression of prostate cancer, cell proliferation, actin cytoskeletal, and actin-related signaling pathways (p < 0.05). Prioritizing genes with vitamin D response elements and associating expression levels with overall survival (OS) in The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA PRAD) cohort, we identified ANLN (Anillin) and ECT2 (Epithelial Cell Transforming 2) as potential prognostic PCa biomarkers. Both genes were strongly correlated and significantly downregulated by 1α,25(OH)2D3 treatment, where low expression was statistically associated with better overall survival outcomes in the TCGA PRAD public cohort. Increased ANLN and ECT2 mRNA gene expression was significantly associated with PCa, and Gleason scores using both the TCGA cohort (p < 0.05) and an AA non-malignant/tumor-matched cohort. Our findings suggest 1α,25(OH)2D3 regulation of these biomarkers may be significant for PCa prevention. In addition, 1α,25(OH)2D3 could be used as an adjuvant treatment targeting actin cytoskeleton signaling and actin cytoskeleton-related signaling pathways, particularly among AA men.

A YTHDF-PABP interaction is required for m6 A-mediated organogenesis in plants.

N6-methyladenosine (m6 A) in mRNA is key to eukaryotic gene regulation. Many m6 A functions involve RNA-binding proteins that recognize m6 A via a YT521-B Homology (YTH) domain. YTH domain proteins contain long intrinsically disordered regions (IDRs) that may mediate phase separation and interaction with protein partners, but whose precise biochemical functions remain largely unknown. The Arabidopsis thaliana YTH domain proteins ECT2, ECT3, and ECT4 accelerate organogenesis through stimulation of cell division in organ primordia. Here, we use ECT2 to reveal molecular underpinnings of this function. We show that stimulation of leaf formation requires the long N-terminal IDR, and we identify two short IDR elements required for ECT2-mediated organogenesis. Of these two, a 19-amino acid region containing a tyrosine-rich motif conserved in both plant and metazoan YTHDF proteins is necessary for binding to the major cytoplasmic poly(A)-binding proteins PAB2, PAB4, and PAB8. Remarkably, overexpression of PAB4 in leaf primordia partially rescues the delayed leaf formation in ect2 ect3 ect4 mutants, suggesting that the ECT2-PAB2/4/8 interaction on target mRNAs of organogenesis-related genes may overcome limiting PAB concentrations in primordial cells.

Evolution of the triplet BRCT domain.

Organisms have evolved a complex system, called the DNA damage response (DDR), which maintains genome integrity. The DDR is responsible for identifying and repairing a variety of lesions and alterations in DNA. DDR proteins coordinate DNA damage detection, cell cycle arrest, and repair, with many of these events regulated by protein phosphorylation. In the human proteome, 23 proteins contain the BRCT (BRCA1 C-Terminus domain) domain, a modular signaling domain that can bind phosphopeptides and mediate protein-protein interactions. BRCTs can be found as functional single units, tandem (tBRCT), triplet (tpBRCT), and quartet. Here we examine the evolution of the tpBRCT architecture present in TOPBP1 (DNA topoisomerase II binding protein 1) and ECT2 (epithelial cell transforming 2), and their respective interaction partners RAD9 (Cell cycle checkpoint control protein RAD9) and CYK-4 (Rac GTPase-activating protein 1), with a focus on the conservation of the phosphopeptide-binding residues. The pair TOPBP1-RAD9 arose with the Eukaryotes and ECT2-CYK-4 with the Eumetazoans. Triplet structural and functional characteristics were conserved in almost all organisms. The first unit of the triplet (BRCT0) is different from the other two BRCTs but conserved between orthologs for both TOPBP1 and ECT2. BRCT domain evolution simulations suggest a trend to retain the singlet or towards two or three BRCT copies per protein consistent with functional tBRCT and tpBRCT architectures. Our results shed light on the emergence of the function and architecture of multiple BRCT domain organizations and provide information about the evolution of the BRCT triplet. Knowledge of BRCT domain evolution can improve the understanding of DNA damage response mechanisms and signal transduction in DDR.

Integration of Chemoinformatics and Multi-Omics Analysis Defines ECT2 as a Potential Target for Cancer Drug Therapy.

Epithelial cell transforming 2 (ECT2) is a potential oncogene and a number of recent studies have correlated it with the progression of several human cancers. Despite this elevated attention for ECT2 in oncology-related reports, there is no collective study to combine and integrate the expression and oncogenic behavior of ECT2 in a panel of human cancers. The current study started with a differential expression analysis of ECT2 in cancerous versus normal tissue. Following that, the study asked for the correlation between ECT2 upregulation and tumor stage, grade, and metastasis, along with its effect on patient survival. Moreover, the methylation and phosphorylation status of ECT2 in tumor versus normal tissue was assessed, in addition to the investigation of the ECT2 effect on the immune cell infiltration in the tumor microenvironment. The current study revealed that ECT2 was upregulated as mRNA and protein levels in a list of human tumors, a feature that allowed for the increased filtration of myeloid-derived suppressor cells (MDSC) and decreased the level of natural killer T (NKT) cells, which ultimately led to a poor prognosis survival. Lastly, we screened for several drugs that could inhibit ECT2 and act as antitumor agents. Collectively, this study nominated ECT2 as a prognostic and immunological biomarker, with reported inhibitors that represent potential antitumor drugs.

m6A readers ECT2/ECT3/ECT4 enhance mRNA stability through direct recruitment of the poly(A) binding proteins in Arabidopsis.

BACKGROUND: RNA N6-methyladenosine (m6A) modification is critical for plant growth and crop yield. m6A reader proteins can recognize m6A modifications to facilitate the functions of m6A in gene regulation. ECT2, ECT3, and ECT4 are m6A readers that are known to redundantly regulate trichome branching and leaf growth, but their molecular functions remain unclear. RESULTS: Here, we show that ECT2, ECT3, and ECT4 directly interact with each other in the cytoplasm and perform genetically redundant functions in abscisic acid (ABA) response regulation during seed germination and post-germination growth. We reveal that ECT2/ECT3/ECT4 promote the stabilization of their targeted m6A-modified mRNAs, but have no function in alternative polyadenylation and translation. We find that ECT2 directly interacts with the poly(A) binding proteins, PAB2 and PAB4, and maintains the stabilization of m6A-modified mRNAs. Disruption of ECT2/ECT3/ECT4 destabilizes mRNAs of ABA signaling-related genes, thereby promoting the accumulation of ABI5 and leading to ABA hypersensitivity. CONCLUSION: Our study reveals a unified functional model of m6A mediated by m6A readers in plants. In this model, ECT2/ECT3/ECT4 promote stabilization of their target mRNAs in the cytoplasm.

Identification and characterization of sex-dependent gene expression profile in glioblastoma.

Glioblastoma (GBM) is the most lethal primary tumor in the human brain and lacks favorable treatment options. Sex differences in the outcome of GBM are broadly acknowledged, but the underlying molecular mechanisms remain largely unknown. To identify the sex-dependent critical genes in the progression of GBM, raw data from several microarray datasets with the same array platform were downloaded from the Gene Expression Omnibus (GEO) database. These datasets included tumorous and normal tissue from patients with GBM and crucial sex features. Then, the differentially expressed genes (DEGs) in female and male tumors were identified via bioinformatics analysis, respectively. Functional signatures of the identified DEGs were further annotated by Gene Ontology (GO) and pathway enrichment analyses. Venn diagram and functional protein-protein interaction (PPI) network analyses were performed to screen out the sex-specific DEGs. Survival analysis of patients with differences in the expression level of selected genes was then carried out using the data from The Cancer Genome Atlas (TCGA). Here, we showed that ECT2, AURKA, TYMS, CDK1, NCAPH, CENPU, OIP5, KIF14, ASPM, FBXO5, SGOL2, CASC5, SHCBP1, FN1, LOX, IGFBP3, CSPG4, and CD44 were enriched in female tumor samples, whereas TNFSF13B, CXCL10, CXCL8, CXCR4, TLR2, CCL2, and FCGR2A were enriched in male tumor samples. Among these key genes, interestingly, ECT2 was associated with increased an survival rate for female patients, whileTNFSF13B could be regarded as a potential marker of poor prognosis in male patients. These results suggested that sex differences in patients may be attributed to the heterogeneous gene activity, which might influence the oncogenesis and the outcomes of GBM.

miRNA-223-3p regulates ECT2 to promote proliferation, invasion, and metastasis of gastric cancer through the Wnt/ß-catenin signaling pathway.

PURPOSE: Expression of the guanine nucleotide exchange factor epithelial cell transforming 2 (ECT2) is elevated in gastric cancer (GC) but its biological function in GC is poorly understood. MicroRNAs (miRNAs) have great potential as therapeutic targets for GC through their ability to modulate gene expression. In the present study, we sought to identify potential miRNA-mRNA-protein regulatory pathways that might control ECT2 expression and function in GC. METHODS: ECT2 expression was examined in clinical GC specimens by immunohistochemical staining, and protein levels were correlated with clinicopathological features and prognosis. TargetScan was used to identify potential ECT2 mRNA-complementary miRNAs, and the roles of ECT2 and miRNA-223-3p (miR-223-3p) in GC cell biology and signaling pathway activation were examined by targeted knockdown (KD) or overexpression (OE) of ECT2 and miR-223-3p in GC cell lines. A murine GC xenograft model was developed to explore the impact of ECT2 OE on tumor growth in vivo. RESULTS: ECT2 expression was significantly elevated in GC specimens compared with normal gastric tissues and the level correlated positively with depth of invasion, ulceration, vascular tumor thrombus, neural invasion, and lymph node metastasis (p < 0.05). ECT2 was an independent prognostic factor for overall survival of GC patients (high ECT2 expression v.s. low ECT2 expression: χ2 = 29.831, p < 0.001). ECT2 KD or miR-223-3p OE markedly suppressed the proliferation, migration, and invasion of GC cells in vitro, whereas ECT2 OE had the opposite effects. ECT2 OE also promoted the growth of GC tumors in vivo. Tumor expression of Wnt2, ß-catenin, and several downstream target proteins in GC cells were decreased by ECT2 KD or miR-223-3p OE but increased by ECT2 OE. CONCLUSIONS: miR-223-3p regulates ECT2 expression to promote tumorigenic behavior of GC via activation of the Wnt/ß-catenin signaling pathway, suggesting that ECT2 and miR-223-3p as potential therapeutic targets for GC.

Long noncoding RNA MAPKAPK5-AS1 promotes metastasis through regulation miR-376b-5p/ECT2 axis in hepatocellular carcinoma.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is one of the most common diseases threatening human health worldwide. However, the molecular mechanisms of HCC are still unclear. Here, we identified a differentially expressed lncRNA called MAPKAPK5-AS1(abbreviation: MK5-AS1) and elucidated its role and molecular mechanism in the development of HCC. METHODS: Real-time PCR (RT-PCR) was used to verify the expression of MK5-AS1 in hepatocarcinoma cell lines and tumor tissues of HCC patients. The biological functions of MK5-AS1 in HCC cells was assessed both in vitro and in vivo assays. The Lncbase, miRDB and TargetScan databases were used to predict the lncRNA-miRNA-mRNA interactions. RNA immunoprecipitation (RIP) and double luciferase reporter gene assays further verified the interactions. RESULTS: MK5-AS1 expression was significantly upregulated in HCC tissues and cell lines. Furthermore, high MK5-AS1 expression was positively associated with tumor progression and poor prognosis. In vitro and in vivo experiments confirmed that overexpressed MK5-AS1 promoted migration and invasion of HCC cells. Bioinformatics analysis based on Lncbase, miRDB and TargetScan databases showed MK5-AS1 competitively bound to miR-376b-5p that prevented epithelial cell transforming sequence 2 (ECT2) from miRNA-mediated degradation, thus facilitated HCC metastasis. CONCLUSION: Our results established a tumor promotive role of MK5-AS1 in HCC pathogenesis.

Increased expression of ECT2 predicts the poor prognosis of breast cancer patients.

Breast cancer is the most common malignancy and the second leading cause of cancer-related death in women. Recent studies have indicated that aberrant activation of Rho GTPases relates to the malignant properties of breast cancer cells. As the guanine nucleotide exchange factor of Rho GTPases, the role of ECT2 (epithelial cell transforming 2) in breast cancer is still unclear. Tissue microarrays and multiple public databases were utilized to investigate the relationship between ECT2 level and clinical-pathological features of breast cancer patients. Kaplan Meier-plotter online tool and tissue microarray with survival information were used to investigate the predictive value for breast cancer. Here, we found increased ECT2 level was highly associated with advanced TNM stage, poor differentiation, and loss of hormone receptors of breast cancer. Gene expression profile showed that ECT2 level was closely correlated to cell-proliferation-associated pathways. Integration analysis using public databases and tissue microarray indicated that high ECT2 was an adverse prognostic factor for breast cancer patients. We believe the ECT2 level might be a valuable complement for commercially available predictors such as the 21 genes test. Furthermore, ECT2 would be a novel target for drug development for breast cancer.

LncRNA FGD5-AS1 enhances the proliferation and stemness of hepatocellular carcinoma cells through targeting miR-223 and regulating the expression of ECT2 and FAT1.

AIM: Hepatocellular carcinoma (HCC) is common and causes many deaths worldwide. The aim of this study is to explore the mechanism by which long non-coding RNA FGD5-AS1 regulates HCC cell proliferation and stemness. METHODS: Tumor and normal adjacent tissues were harvested from HCC patients. Real-time quantitative reverse transcription-PCR was applied to examine the expression of FGD5-AS1, miR-223, Epithelial cell transforming sequence 2 (ECT2) and FAT1. The protein levels of ECT2, FAT1, proliferating cell nuclear antigen (PCNA), OCT4, CD133 and CD90 were analyzed by western blot. The localization of FGD5-AS1 was examined by Fluorescence in situ hybridization. Cell proliferation was analyzed with CCK-8 and colony formation assays. Spheroid formation was used for analyzing cell stemness. Gene interaction was examined by RNA immunoprecipitation and luciferase activity assays. A subcutaneous xenograft mouse model was established to analyze HCC growth and stemness in vivo. Immunohistochemistry staining was used to analyze the expression PCNA and OCT4 in subcutaneous tumors. RESULTS: FGD5-AS1 was upregulated in HCC and its high expression indicated poor prognosis of patients. High expression of FGD5-AS1 enhanced HCC cell proliferation and stemness. Knockdown of FGD5-AS1 restrained tumor growth and stemness in mice. FGD5-AS1 directly sponged miR-223 and promoted the expression of ECT2 and FAT1 in HCC. Both knockdown of miR-223 and overexpression of ECT2 and FAT1 reversed FGD5-AS1 silencing-mediated suppression of HCC cell proliferation and stemness. CONCLUSION: FGD5-AS1 directly sponged miR-223 and promoted the expression of ECT2 and FAT1 in HCC, thus enhancing HCC cell proliferation and stemness. Our study identifies potential prognostic biomarkers and therapeutic targets for HCC.

An update on principles of m6A targeting.

N6-methyladenosine (m6A) is of fundamental importance in gene regulation. The function of m6A is achieved through proteins that recognize m6A, known as EVOLUTIONARILY CONSERVED C-TERMINAL REGIONS (ECTs) in arabidopsis (Arabidopsis thaliana). mRNA targets of ECTs and their interaction with m6A-containing motifs remain to be revealed. In this forum article, I highlight recent advances in m6A targeting.

The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation.

Gene regulation via N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only a few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant, ectopic, and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2, ECT3, and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady-state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.

Principles of mRNA targeting via the Arabidopsis m6A-binding protein ECT2.

Specific recognition of N6-methyladenosine (m6A) in mRNA by RNA-binding proteins containing a YT521-B homology (YTH) domain is important in eukaryotic gene regulation. The Arabidopsis YTH domain protein ECT2 is thought to bind to mRNA at URU(m6A)Y sites, yet RR(m6A)CH is the canonical m6A consensus site in all eukaryotes and ECT2 functions require m6A-binding activity. Here, we apply iCLIP (individual nucleotide resolution crosslinking and immunoprecipitation) and HyperTRIBE (targets of RNA-binding proteins identified by editing) to define high-quality target sets of ECT2 and analyze the patterns of enriched sequence motifs around ECT2 crosslink sites. Our analyses show that ECT2 does in fact bind to RR(m6A)CH. Pyrimidine-rich motifs are enriched around, but not at m6A sites, reflecting a preference for N6-adenosine methylation of RRACH/GGAU islands in pyrimidine-rich regions. Such motifs, particularly oligo-U and UNUNU upstream of m6A sites, are also implicated in ECT2 binding via its intrinsically disordered region (IDR). Finally, URUAY-type motifs are enriched at ECT2 crosslink sites, but their distinct properties suggest function as sites of competition between binding of ECT2 and as yet unidentified RNA-binding proteins. Our study provides coherence between genetic and molecular studies of m6A-YTH function in plants and reveals new insight into the mode of RNA recognition by YTH domain-containing proteins.

Genes are strings of genetic code that contain instructions for producing a cell's proteins. Active genes are copied from DNA into molecules called mRNAs, and mRNA molecules are subsequently translated to create new proteins. However, the number of proteins produced by a cell is not only limited by the number of mRNA molecules produced by copying DNA. Cells use a variety of methods to control the stability of mRNA molecules and their translation efficiency to regulate protein production. One of these methods involves adding a chemical tag, a methyl group, onto mRNA while it is being created. These methyl tags can then be used as docking stations by RNA-binding proteins that help regulate protein translation. Most eukaryotic species ­ which include animals, plants and fungi ­ use the same system to add methyl tags to mRNA molecules. One methyl tag in particular, known as m⁶A, is a well-characterised docking site for a particular type of RNA-binding protein that goes by the name of ECT2 in plants. However, in the flowering plant Arabidopsis thaliana, ECT2 was thought to bind to an mRNA sequence different from the one normally carrying the chemical tag, creating obvious confusion about how the system works in plants. Arribas-Hernández, Rennie et al. investigated this question using advanced large-scale biochemical techniques, and discovered that conventional m⁶A methyl tags are indeed used by ECT2 in Arabidopsis thaliana. The confusion likely arose because the sequence ECT2 was thought bind is often located in close proximity to the m⁶A tags, possibly acting as docking stations for proteins that can influence the ability of ECT2 to bind mRNA. Arribas-Hernández, Rennie et al. also uncovered additional mRNA sequences that directly interact with parts of ECT2 previously unknown to participate in mRNA binding. These findings provide new insights into how chemical labels in mRNA control gene activity. They have broad implications that extend beyond plants into other eukaryotic species, including humans. Since this chemical labelling system has a major role in controlling plant growth, these findings could be leveraged in biotechnology applications to improve crop yields and enhance plant-based food production.

Phosphorylation of ß-catenin Ser60 by polo-like kinase 1 drives the completion of cytokinesis.

ß-Catenin is a multifunctional protein and participates in numerous processes required for embryonic development, cell proliferation, and homeostasis through various molecular interactions and signaling pathways. To date, however, there is no direct evidence that ß-catenin contributes to cytokinesis. Here, we identify a novel p-S60 epitope on ß-catenin generated by Plk1 kinase activity, which can be found at the actomyosin contractile ring of early telophase cells and at the midbody of late telophase cells. Depletion of ß-catenin leads to cytokinesis-defective phenotypes, which eventually result in apoptotic cell death. In addition, phosphorylation of ß-catenin Ser60 by Plk1 is essential for the recruitment of Ect2 to the midbody, activation of RhoA, and interaction between ß-catenin, Plk1, and Ect2. Time-lapse image analysis confirmed the importance of ß-catenin phospho-Ser60 in furrow ingression and the completion of cytokinesis. Taken together, we propose that phosphorylation of ß-catenin Ser60 by Plk1 in cooperation with Ect2 is essential for the completion of cytokinesis. These findings may provide fundamental knowledge for the research of cytokinesis failure-derived human diseases.

Alveolar epithelial cell fate is maintained in a spatially restricted manner to promote lung regeneration after acute injury.

Alveolar epithelial type 2 (AT2) cells integrate signals from multiple molecular pathways to proliferate and differentiate to drive regeneration of the lung alveolus. Utilizing in vivo genetic and ex vivo organoid models, we investigated the role of Fgfr2 signaling in AT2 cells across the lifespan and during adult regeneration after influenza infection. We show that, although dispensable for adult homeostasis, Fgfr2 restricts AT2 cell fate during postnatal lung development. Using an unbiased computational imaging approach, we demonstrate that Fgfr2 promotes AT2 cell proliferation and restrains differentiation in actively regenerating areas after injury. Organoid assays reveal that Fgfr2-deficient AT2 cells remain competent to respond to multiple parallel proliferative inputs. Moreover, genetic blockade of AT2 cell cytokinesis demonstrates that cell division and differentiation are uncoupled during alveolar regeneration. These data reveal that Fgfr2 maintains AT2 cell fate, balancing proliferation and differentiation during lung alveolar regeneration.

High expression of Ras-specific guanine nucleotide-releasing factor 2 (RasGRF2) in lung adenocarcinoma is associated with tumor invasion and poor prognosis.

The expression of Ras-specific guanine nucleotide-releasing factor 2 (RasGRF2) in lung adenocarcinomas was examined using immunohistochemistry in relation to clinicopathological characteristics and prognosis. In comparison to low expression, high expression of RasGRF2 was more closely associated with poor prognosis. Interestingly, expression of phosphorylated epithelial cell transforming 2 (pECT2), which - like RasGRF2 - is also a guanine-nucleotide exchange factor, was also associated with prognosis, and patients with high expression of both RasGRF2 and pECT2 had a much poorer outcome than those who were negative for both.

The BRCT domains of ECT2 have distinct functions during cytokinesis.

During cell division, the guanine nucleotide exchange factor (GEF) ECT2 activates RhoA in a narrow zone at the cell equator in anaphase. ECT2 consists of three BRCT domains (BRCT0, 1, and 2), a catalytic GEF, and a pleckstrin homology (PH) domain. How the conserved BRCT domains spatially and temporally control ECT2 activity remains unclear. We reveal that each BRCT domain makes distinct contributions to the ECT2 function. We find that BRCT0 contributes to, and BRCT1 is essential for, ECT2 activation in anaphase. BRCT2 integrates two functions: GEF inhibition and RACGAP1 binding, which together limit ECT2 activity to a narrow zone at the cell equator. BRCT2-dependent control of active RhoA zone dimension functions in addition to the inhibitory signal of the astral microtubules. Our analysis provides detailed mechanistic insights into how ECT2 activity is regulated and how that regulation ensures, together with other signaling pathways, successful cell division.

Role of Small GTPase RhoA in DNA Damage Response.

Accumulating evidence has suggested a role of the small GTPase Ras homolog gene family member A (RhoA) in DNA damage response (DDR) in addition to its traditional function of regulating cell morphology. In DDR, 2 key components of DNA repair, ataxia telangiectasia-mutated (ATM) and flap structure-specific endonuclease 1 (FEN1), along with intracellular reactive oxygen species (ROS) have been shown to regulate RhoA activation. In addition, Rho-specific guanine exchange factors (GEFs), neuroepithelial transforming gene 1 (Net1) and epithelial cell transforming sequence 2 (Ect2), have specific functions in DDR, and they also participate in Ras-related C3 botulinum toxin substrate 1 (Rac1)/RhoA interaction, a process which is largely unappreciated yet possibly of significance in DDR. Downstream of RhoA, current evidence has highlighted its role in mediating cell cycle arrest, which is an important step in DNA repair. Unraveling the mechanism by which RhoA modulates DDR may provide more insight into DDR itself and may aid in the future development of cancer therapies.