SMU1 Knockdown Suppresses Gastric Carcinoma Growth, Migration, and Invasion and Modulates the Cell Cycle.

INTRODUCTION: The role of SMU1 in DNA replication and RNA splicing is well-established, yet its specific function and dysregulated mechanisms in gastric cancer (GC) remain inadequately elucidated. This study seeks to investigate the potential oncogenic and progression-promoting effects of SMU1 in GC, with the ultimate goal of informing novel approaches for treatment and diagnosis. METHODS: The study investigated the expression levels of SMU1 in GC and adjacent normal tissues by analyzing data from the TCGA (27 tissue pairs) and GEO (47 tissue pairs) databases. Immunohistochemistry was used to examine 277 tumor tissue and adjacent non-tumor tissue spots from GC tissue chips, along with relevant follow-up information. The study further assessed the proliferation, invasion, and migration capabilities of cells by manipulating SMU1 expression levels and conducting various assays, including CCK-8, EdU incorporation, colony formation, transwells, flow cytometry, and subcutaneous tumorigenesis assays. RESULTS: Our study revealed a significant upregulation of SMU1 mRNA and protein levels in GC tissues compared to adjacent tissues. Univariate and multivariate Cox analysis demonstrated that elevated levels of SMU1 were independent prognostic factors for GC prognosis (P = 0.036). Additionally, median survival analysis indicated a significant association between high SMU1 expression and poor prognosis in GC patients (P = 0.0002). In experiments conducted both in vivo and in vitro, it was determined that elevated levels of SMU1 can enhance the proliferation, invasion, and migration of GC cells, whereas suppression of SMU1 can impede the progression of GC by modulating the G1/S checkpoint of the cell cycle. CONCLUSIONS: Our research introduces the novel idea that SMU1 could serve as a prognostic marker for GC progression, influencing cell proliferation through cell cycle activation. These results offer valuable insights into the understanding, diagnosis, and management of gastric carcinoma.

NCAPD2 serves as a potential prognostic biomarker for lung adenocarcinoma and promotes cell proliferation, migration, invasion and cell cycle in vitro.

Objectives: The pro-oncogenic effects of NCAPD2 have been extensively studied across various tumor types; however, its precise role within the context of lung adenocarcinoma (LUAD) remains elusive. This study aims to elucidate the biological functions of NCAPD2 in LUAD and unravel the underlying mechanistic pathways. Methods: Utilizing bioinformatics methodologies, we explored the differential expression of NCAPD2 between normal and tumor samples, along with its correlations with clinical-pathological characteristics, survival prognosis, and immune infiltration. Results: In the TCGA-LUAD dataset, tumor samples demonstrated significantly elevated levels of NCAPD2 expression compared to normal samples (p < 0.001). Clinically, higher NCAPD2 expression was notably associated with advanced T, N, and M stages, pathologic stage, gender, smoking status, and diminished overall survival (OS). Moreover, differentially expressed genes (DEGs) associated with NCAPD2 were predominantly enriched in pathways related to cell division. Immune infiltration analysis revealed that NCAPD2 expression levels were linked to the infiltration of memory B cells, naïve CD4+ T cells, activated memory CD4+ T cells, and M1 macrophages. In vitro experiments demonstrated that silencing NCAPD2 suppressed LUAD cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and cell cycle progression. Conclusions: In summary, NCAPD2 may represent a promising prognostic biomarker and novel therapeutic target for LUAD.

Case Report: Molecular Analyses of Cell-Cycle-Related Genes in Cortical Brain Tissue of a Patient with Rasmussen Encephalitis.

Rasmussen's encephalitis (RE) stands as a rare neurological disorder marked by progressive cerebral hemiatrophy and epilepsy resistant to medical treatment. Despite extensive study, the primary cause of RE remains elusive, while its histopathological features encompass cortical inflammation, neuronal degeneration, and gliosis. The underlying molecular mechanisms driving disease progression remain largely unexplored. In this case study, we present a patient with RE who underwent hemispherotomy and has remained seizure-free for over six months, experiencing gradual motor improvement. Furthermore, we conducted molecular analysis on the excised brain tissue, unveiling a decrease in the expression of cell-cycle-associated genes coupled with elevated levels of BDNF and TNF-α proteins. These findings suggest the potential involvement of cell cycle regulators in the progression of RE.

Global transcription regulation revealed from dynamical correlations in time-resolved single-cell RNA sequencing.

Single-cell transcriptomics reveals significant variations in transcriptional activity across cells. Yet, it remains challenging to identify mechanisms of transcription dynamics from static snapshots. It is thus still unknown what drives global transcription dynamics in single cells. We present a stochastic model of gene expression with cell size- and cell cycle-dependent rates in growing and dividing cells that harnesses temporal dimensions of single-cell RNA sequencing through metabolic labeling protocols and cel lcycle reporters. We develop a parallel and highly scalable approximate Bayesian computation method that corrects for technical variation and accurately quantifies absolute burst frequency, burst size, and degradation rate along the cell cycle at a transcriptome-wide scale. Using Bayesian model selection, we reveal scaling between transcription rates and cell size and unveil waves of gene regulation across the cell cycle-dependent transcriptome. Our study shows that stochastic modeling of dynamical correlations identifies global mechanisms of transcription regulation. A record of this paper's transparent peer review process is included in the supplemental information.

High Glycolytic Activity Signature Reveals CCNB2 as a Key Therapeutic Target in Triple-Negative Breast Cancer.

BACKGROUND: Aerobic glycolysis and the cell cycle are well-established tumor hallmarks. Understanding their relationship could help to unravel the pathogenic mechanisms of breast cancer (BC) and suggest potential new strategies for treatment. METHODS: Glycolysis-related genes (GRGs) were downloaded from the Reactome database and screened using univariate Cox analysis. The consensus clustering method was employed to identify a glycolytic activity signature (GAS) using the Gene Expression Omnibus (GEO) dataset. A nomogram risk prediction model was constructed using coefficients from univariate Cox analysis. Immune cell infiltration was evaluated using single-sample gene set enrichment analysis (ssGSEA) and the ESTIMATE algorithm. Gene co-expression modules were created using weighted correlation network analysis (WGCNA) to identify hub genes. Gene expression in three BC cell lines was quantified using Quantitative Reverse Transcriptase Polymera (qRT-PCR). Single-cell RNA sequencing (scRNA-seq) data was used to examine the relationship between GAS and hub genes. The sensitivity of different groups to cell cycle-related clinical drugs was also examined. RESULTS: BC with high GAS (HGAS) showed high tumor grade and recurrence rate. HGAS was a prognostic indicator of worse overall survival (OS) in BC patients. HGAS BC showed more abundant immune cells and significantly higher expression of immunomodulators compared to BC with low GAS (LGAS). HGAS BC also showed enhanced cell cycle pathway, with high mRNA and protein expression levels of Cyclin B2 (CCNB2), a key component of the cell cycle pathway. Importantly, scRNA-seq analysis revealed that elevated CCNB2 expression was positively correlated with HGAS in triple-negative BC (TNBC). This was validated in clinical samples from TNBC patients. High expression of CCNB2 was found in three BC cell lines, and was also an indicator of poor prognosis. HGAS BC showed high sensitivity to several cell cycle-related clinical drugs, with 9 of these also showing activity in BC with high CCNB2 expression. CONCLUSIONS: HGAS was associated with enhanced cell cycle pathway and immune activity in BC. These results suggest that CCNB2 is a potential key therapeutic target in BC patients.

Clinical pathological significance and biological function of PLIN1 in hepatocellular carcinoma: bioinformatics analysis and in vitro experiments.

BACKGROUND & AIMS: Perilipin 1 (PLIN1) is an essential lipid droplet surface protein that participates in cell life activities by regulating energy balance and lipid metabolism. PLIN1 has been shown to be closely related to the development of numerous tumor types. The purpose of this work was to elucidate the clinicopathologic significance of PLIN1 in hepatocellular carcinoma (HCC), as well as its impact on the biological functions of HCC cells, and to investigate the underlying mechanisms involved. METHODS: Public high-throughput RNA microarray and RNA sequencing data were collected to examine PLIN1 levels and clinical significance in patients with HCC. Immunohistochemistry (IHC) and real-time quantitative reverse transcription polymerase chain reaction (RT‒qPCR) were conducted to assess the expression levels and the clinicopathological relevance of PLIN1 in HCC. Then, SK and Huh7 cells were transfected with a lentivirus overexpressing PLIN1. CCK8 assay, wound healing assay, transwell assay, and flow cytometric analysis were conducted to explore the effects of PLIN1 overexpression on HCC cell proliferation, migration, invasion, and cell cycle distribution. Ultimately, Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to investigate the underlying mechanisms of PLIN1 in HCC progression based on HCC differentially expressed genes and PLIN1 co-expressed genes. RESULTS: PLIN1 was markedly downregulated in HCC tissues, which correlated with a noticeably worse prognosis for HCC patients. Additionally, PLIN1 overexpression inhibited the proliferation, migration, and invasion of SK and Huh7 cells in vitro, as well as arresting the HCC cell cycle at the G0/G1 phase. More significantly, energy conversion-related biological processes, lipid metabolism, and cell cycle signalling pathways were the three most enriched molecular mechanisms. CONCLUSION: The present study revealed that PLIN1 downregulation is associated with poor prognosis in HCC patients and accelerated HCC progression by promoting cellular proliferation, migration, and metastasis, as well as the mechanisms underlying the regulation of lipid metabolism-related pathways in HCC.

MANCR lncRNA Modulates Cell-Cycle Progression and Metastasis by Cis-Regulation of Nuclear Rho-GEF.

A significant number of the genetic alterations observed in cancer patients lie within nonprotein-coding segments of the genome, including regions coding for long noncoding RNAs (lncRNAs). LncRNAs display aberrant expression in breast cancer (BrCa), but the functional implications of this altered expression remain to be elucidated. By performing transcriptome screen in a triple negative BrCa (TNBC) isogenic 2D and 3D spheroid model, we observed aberrant expression of >1000 lncRNAs during BrCa progression. The chromatin-associated lncRNA MANCR shows elevated expression in metastatic TNBC. MANCR is upregulated in response to cellular stress and modulates DNA repair and cell proliferation. MANCR promotes metastasis as MANCR-depleted cells show reduced cell migration, invasion, and wound healing in vitro, and reduced metastatic lung colonization in xenograft experiments in vivo. Transcriptome analyses reveal that MANCR modulates expression and pre-mRNA splicing of genes, controlling DNA repair and checkpoint response. MANCR promotes the transcription of NET1A, a Rho-GEF that regulates DNA damage checkpoint and metastatic processes in cis, by differential promoter usage. Experiments suggest that MANCR regulates the expression of cancer-associated genes by modulating the association of various transcription factors and RNA-binding proteins. Our results identified the metastasis-promoting activities of MANCR in TNBC by cis-regulation of gene expression.

Single cell expression and chromatin accessibility of the Toxoplasma gondii lytic cycle identifies AP2XII-8 as an essential ribosome regulon driver.

Sequential lytic cycles driven by cascading transcriptional waves underlie pathogenesis in the apicomplexan parasite Toxoplasma gondii. This parasite's unique division by internal budding, short cell cycle, and jumbled up classically defined cell cycle stages have restrained in-depth transcriptional program analysis. Here, unbiased transcriptome and chromatin accessibility maps throughout the lytic cell cycle are established at the single-cell level. Correlated pseudo-timeline assemblies of expression and chromatin profiles maps transcriptional versus chromatin level transition points promoting the cell division cycle. Sequential clustering analysis identifies functionally related gene groups promoting cell cycle progression. Promoter DNA motif mapping reveals patterns of combinatorial regulation. Pseudo-time trajectory analysis reveals transcriptional bursts at different cell cycle points. The dominant burst in G1 is driven largely by transcription factor AP2XII-8, which engages a conserved DNA motif, and promotes the expression of 44 ribosomal proteins encoding regulon. Overall, the study provides integrated, multi-level insights into apicomplexan transcriptional regulation.

Mitochondrial double-stranded RNA homeostasis depends on cell-cycle progression.

Mitochondrial gene expression is a compartmentalised process essential for metabolic function. The replication and transcription of mitochondrial DNA (mtDNA) take place at nucleoids, whereas the subsequent processing and maturation of mitochondrial RNA (mtRNA) and mitoribosome assembly are localised to mitochondrial RNA granules. The bidirectional transcription of circular mtDNA can lead to the hybridisation of polycistronic transcripts and the formation of immunogenic mitochondrial double-stranded RNA (mt-dsRNA). However, the mechanisms that regulate mt-dsRNA localisation and homeostasis are largely unknown. With super-resolution microscopy, we show that mt-dsRNA overlaps with the RNA core and associated proteins of mitochondrial RNA granules but not nucleoids. Mt-dsRNA foci accumulate upon the stimulation of cell proliferation and their abundance depends on mitochondrial ribonucleotide supply by the nucleoside diphosphate kinase, NME6. Consequently, mt-dsRNA foci are profuse in cultured cancer cells and malignant cells of human tumour biopsies. Our results establish a new link between cell proliferation and mitochondrial nucleic acid homeostasis.

Expression and prognostic value of cell-cycle-associated genes in lung squamous cell carcinoma.

BACKGROUND: Lung cancer continues to be a prevalent cause of cancer-related deaths worldwide, with lung squamous carcinoma (LUSC) being a significant subtype characterized by comparatively low survival rates. Extensive molecular studies on LUSC have been conducted; however, the clinical importance of cell-cycle-associated genes has rarely been examined. This study aimed to investigate the relationship between these genes and LUSC. METHODS: The expression trends of genes related to the cell cycle in a group of patients with LUSC were analyzed. Clinical information and mRNA expression data were obtained from The Cancer Genome Atlas via cBioportal. Multiple analyses have been performed to investigate the association between these genes and LUSC. RESULTS: Three clusters were identified based on the mRNA expression of 124 cell cycle-associated genes. Cluster 3 exhibited the worst prognosis. A comparative analysis showed that nine expressed genes differed distinctly among all the clusters. Among these nine genes, elevated expression of CDK4 was strongly associated with positive prognosis. Furthermore, the expression of ANAPC11, ANAPC5, and ORC4 correlated with the advancement of LUSC pathological stages. CONCLUSIONS: Gene expression profiles associated with the cell cycle across various LUSC subtypes were identified, highlighting that specific genes are related to prognosis and disease stages. Based on these results, new prognostic strategies, patient stratification, and targeted therapy trials have been conducted for LUSC.

Hominini-specific regulation of the cell cycle by stop codon readthrough of FEM1B.

FEM1B is a substrate-recognition component of the CRL2 E3 ubiquitin-protein ligase. This multi-protein complex targets specific proteins for ubiquitylation, which leads to their degradation. Here, we demonstrate the regulation of FEM1B expression by stop codon readthrough (SCR). In this process, translating ribosomes readthrough the stop codon of FEM1B to generate a C-terminally extended isoform that is highly unstable. A total of 81 nucleotides in the proximal 3'UTR of FEM1B constitute the necessary and sufficient cis-signal for SCR. Also, they encode the amino acid sequence responsible for the degradation of the SCR product. CRISPR-edited cells lacking this region, and therefore SCR of FEM1B, showed increased FEM1B expression. This in turn resulted in reduced expression of SLBP (a target of FEM1B-mediated degradation) and replication-dependent histones (target of SLBP for mRNA stability), causing cell cycle delay. Evolutionary analysis revealed that this phenomenon is specific to the genus Pan and Homo (Hominini). Overall, we show a relatively recently evolved SCR process that relieves the cell cycle from the negative regulation by FEM1B.

Exploring the clinical and biological significance of the cell cycle-related gene CHMP4C in prostate cancer.

BACKGROUND: Prostate cancer (PCa) stands as the second most prevalent malignancy impacting male health, and the disease's evolutionary course presents formidable challenges in the context of patient treatment and prognostic management. Charged multivesicular body protein 4 C (CHMP4C) participates in the development of several cancers by regulating cell cycle functions. However, the role of CHMP4C in prostate cancer remains unclear. METHODS: In terms of bioinformatics, multiple PCa datasets were employed to scrutinize the expression of CHMP4C. Survival analysis coupled with a nomogram approach was employed to probe into the prognostic significance of CHMP4C. Gene set enrichment analysis (GSEA) was conducted to interrogate the functional implications of CHMP4C. In terms of cellular experimentation, the verification of RNA and protein expression levels was executed through the utilization of qRT-PCR and Western blotting. Upon the establishment of a cell line featuring stable CHMP4C knockdown, a battery of assays, including Cell Counting Kit-8 (CCK-8), wound healing, Transwell, and flow cytometry, were employed to discern the impact of CHMP4C on the proliferation, migration, invasion, and cell cycle function of PCa cells. RESULTS: The expression of CHMP4C exhibited upregulation in both PCa cells and tissues, and patients demonstrating elevated CHMP4C expression levels experienced a notably inferior prognosis. The nomogram, constructed using CHMP4C along with clinicopathological features, demonstrated a commendable capacity for prognostic prediction. CHMP4C knockdown significantly inhibited the proliferation, migration, and invasion of PCa cells (LNcaP and PC3). CHMP4C could impact the advancement of the PCa cell cycle, and its expression might be regulated by berberine. Divergent CHMP4C expression among PCa patients could induce alterations in immune cell infiltration and gene mutation frequency. CONCLUSIONS: Our findings suggest that CHMP4C might be a prognostic biomarker in PCa, potentially offering novel perspectives for the advancement of precision therapy for PCa.

A nucleic acid binding protein map of germline regulation in Caenorhabditis elegans.

Fertility requires the faithful proliferation of germ cells and their differentiation into gametes. Controlling these cellular states demands precise timing and expression of gene networks. Nucleic acid binding proteins (NBPs) play critical roles in gene expression networks that influence germ cell development. There has, however, been no functional analysis of the entire NBP repertoire in controlling in vivo germ cell development. Here, we analyzed germ cell states and germline architecture to systematically investigate the function of 364 germline-expressed NBPs in the Caenorhabditis elegans germ line. Using germline-specific knockdown, automated germ cell counting, and high-content analysis of germ cell nuclei and plasma membrane organization, we identify 156 NBPs with discrete autonomous germline functions. By identifying NBPs that control the germ cell cycle, proliferation, differentiation, germline structure and fertility, we have created an atlas for mechanistic dissection of germ cell behavior and gamete production.

Single-cell RNA sequencing reveals placental response under environmental stress.

The placenta is crucial for fetal development, yet the impact of environmental stressors such as arsenic exposure remains poorly understood. We apply single-cell RNA sequencing to analyze the response of the mouse placenta to arsenic, revealing cell-type-specific gene expression, function, and pathological changes. Notably, the Prap1 gene, which encodes proline-rich acidic protein 1 (PRAP1), is significantly upregulated in 26 placental cell types including various trophoblast cells. Our study shows a female-biased increase in PRAP1 in response to arsenic and localizes it in the placenta. In vitro and ex vivo experiments confirm PRAP1 upregulation following arsenic treatment and demonstrate that recombinant PRAP1 protein reduces arsenic-induced cytotoxicity and downregulates cell cycle pathways in human trophoblast cells. Moreover, PRAP1 knockdown differentially affects cell cycle processes, proliferation, and cell death depending on the presence of arsenic. Our findings provide insights into the placental response to environmental stress, offering potential preventative and therapeutic approaches for environment-related adverse outcomes in mothers and children.

Cyclin F-EXO1 axis controls cell cycle-dependent execution of double-strand break repair.

Ubiquitination is a crucial posttranslational modification required for the proper repair of DNA double-strand breaks (DSBs) induced by ionizing radiation (IR). DSBs are mainly repaired through homologous recombination (HR) when template DNA is present and nonhomologous end joining (NHEJ) in its absence. In addition, microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA) provide backup DSBs repair pathways. However, the mechanisms controlling their use remain poorly understood. By using a high-resolution CRISPR screen of the ubiquitin system after IR, we systematically uncover genes required for cell survival and elucidate a critical role of the E3 ubiquitin ligase SCFcyclin F in cell cycle-dependent DSB repair. We show that SCFcyclin F-mediated EXO1 degradation prevents DNA end resection in mitosis, allowing MMEJ to take place. Moreover, we identify a conserved cyclin F recognition motif, distinct from the one used by other cyclins, with broad implications in cyclin specificity for cell cycle control.

Doxorubicin downregulates cell cycle regulatory hub genes in breast cancer cells.

Breast cancer (BC) is the leading commonly diagnosed cancer in the world, with complex mechanisms underlying its development. There is an urgent need to enlighten key genes as potential therapeutic targets crucial to advancing BC treatment. This study sought to investigate the influence of doxorubicin (DOX) on identified key genes consistent across numerous BC datasets obtained through bioinformatic analysis. To date, a meta-analysis of publicly available coding datasets for expression profiling by array from the Gene Expression Omnibus (GEO) has been carried out. Differentially Expressed Genes (DEGs) identified using GEO2R revealed a total of 23 common DEGs, including nine upregulated genes and 14 downregulated genes among the datasets of three platforms (GPL570, GPL6244, and GPL17586), and the commonly upregulated DEGs, showed significant enrichment in the cell cycle in KEGG analysis. The top nine genes, NUSAP1, CENPF, TPX2, PRC1, ANLN, BUB1B, AURKA, CCNB2, and CDK-1, with higher degree values and MCODE scores in the cytoscape program, were regarded as hub genes. The hub genes were activated in disease states commonly across all the subclasses of BC and correlated with the unfavorable overall survival of BC patients, as verified by the GEPIA and UALCAN databases. qRT-PCR confirmed that DOX treatment resulted in reduced expression of these genes in BC cell lines, which reinforces the evidence that DOX remains an effective drug for BC and suggests that developing modified formulations of doxorubicin to reduce toxicity and resistance, could enhance its efficacy as an effective therapeutic option for BC.

CRISPR/Cas-Mediated Knockdown of PD-L1 and KRAS in Lung Cancer Cells.

Cancer cells can escape death and surveillance by the host immune system in various ways. Programmed cell death ligand 1 (PD-L1) is a transmembrane protein that is expressed by most cell types, including cancer cells, and can provide an inhibitory signal to its receptor PD-1, which is expressed on the surface of activated T cells, impairing the immune response. PD-L1/PD-1-mediated immune evasion is observed in several KRAS-mutated cancers. In the current study, we used the CRISPR/Cas9 system to knock down PD-L1 and KRAS in adenocarcinoma lung cells (A549 and H1975). Knockdown of PD-L1 was validated by qPCR and coculture with lymphocytes. The cells were functionally analyzed for cell cycle, migration and apoptosis. In addition, the effects of PD-L1 and KRAS downregulation on chemotherapy sensitivity and expression of inflammatory markers were investigated. Suppression of PD-L1 and KRAS led to a slowdown of the cell cycle in the G0/G1 phase and reduced migration, increased sensitivity to chemotherapy and triggered apoptosis of cancer cells. In addition, the conditioned medium of the modulated cells significantly affected the native cancer cells and reduced their viability and drug resistance. Our study suggests that dual silencing of PD-L1 and KRAS by CRISPR/Cas9 may be a promising therapeutic approach for the treatment of lung cancer.

The Escherichia coli chromosome moves to the replisome.

In Escherichia coli, it is debated whether the two replisomes move independently along the two chromosome arms during replication or if they remain spatially confined. Here, we use high-throughput fluorescence microscopy to simultaneously determine the location and short-time-scale (1 s) movement of the replisome and a chromosomal locus throughout the cell cycle. The assay is performed for several loci. We find that (i) the two replisomes are confined to a region of ~250 nm and ~120 nm along the cell's long and short axis, respectively, (ii) the chromosomal loci move to and through this region sequentially based on their distance from the origin of replication, and (iii) when a locus is being replicated, its short time-scale movement slows down. This behavior is the same at different growth rates. In conclusion, our data supports a model with DNA moving towards spatially confined replisomes at replication.

Regulators of rDNA array morphology in fission yeast.

Nucleolar morphology is a well-established indicator of ribosome biogenesis activity that has served as the foundation of many screens investigating ribosome production. Missing from this field of study is a broad-scale investigation of the regulation of ribosomal DNA morphology, despite the essential role of rRNA gene transcription in modulating ribosome output. We hypothesized that the morphology of rDNA arrays reflects ribosome biogenesis activity. We established GapR-GFP, a prokaryotic DNA-binding protein that recognizes transcriptionally-induced overtwisted DNA, as a live visual fluorescent marker for quantitative analysis of rDNA organization in Schizosaccharomyces pombe. We found that the morphology-which we refer to as spatial organization-of the rDNA arrays is dynamic throughout the cell cycle, under glucose starvation, RNA pol I inhibition, and TOR activation. Screening the haploid S. pombe Bioneer deletion collection for spatial organization phenotypes revealed large ribosomal protein (RPL) gene deletions that alter rDNA organization. Further work revealed RPL gene deletion mutants with altered rDNA organization also demonstrate resistance to the TOR inhibitor Torin1. A genetic analysis of signaling pathways essential for this resistance phenotype implicated many factors including a conserved MAPK, Pmk1, previously linked to extracellular stress responses. We propose RPL gene deletion triggers altered rDNA morphology due to compensatory changes in ribosome biogenesis via multiple signaling pathways, and we further suggest compensatory responses may contribute to human diseases such as ribosomopathies. Altogether, GapR-GFP is a powerful tool for live visual reporting on rDNA morphology under myriad conditions.

Direct single-cell observation of a key Escherichia coli cell-cycle oscillator.

Initiation of DNA replication in Escherichia coli is coupled to cell size via the DnaA protein, whose activity is dependent on its nucleotide-bound state. However, the oscillations in DnaA activity have never been observed at the single-cell level. By measuring the volume-specific production rate of a reporter protein under control of a DnaA-regulated promoter, we could distinguish two distinct cell-cycle oscillators. The first, driven by both DnaA activity and SeqA repression, shows a causal relationship with cell size and divisions, similarly to initiation events. The second one, a reporter of DnaA activity alone, loses the synchrony and causality properties. Our results show that transient inhibition of gene expression by SeqA keeps the oscillation of volume-sensing DnaA activity in phase with the subsequent division event and suggest that DnaA activity peaks do not correspond directly to initiation events.