SCAMP4 enhances the senescent cell secretome.

The senescence-associated secretory phenotype (SASP) is a major trait of senescent cells, but the molecular regulators of SASP factor secretion are poorly understood. Mass spectrometry analysis revealed that secretory carrier membrane protein 4 (SCAMP4) levels were strikingly elevated on the surface of senescent cells compared with proliferating cells. Interestingly, silencing SCAMP4 in senescent fibroblasts reduced the secretion of SASP factors, including interleukin 6 (IL6), IL8, growth differentiation factor 15 (GDF-15), C-X-C motif chemokine ligand 1 (CXCL1), and IL7, while, conversely, SCAMP4 overexpression in proliferating fibroblasts increased SASP factor secretion. Our results indicate that SCAMP4 accumulates on the surface of senescent cells, promotes SASP factor secretion, and critically enhances the SASP phenotype.

Identification of senescent cell surface targetable protein DPP4.

Senescent cell accumulation in aging tissues is linked to age-associated diseases and declining function, prompting efforts to eliminate them. Mass spectrometry analysis revealed that DPP4 (dipeptidyl peptidase 4) was selectively expressed on the surface of senescent, but not proliferating, human diploid fibroblasts. Importantly, the differential presence of DPP4 allowed flow cytometry-mediated isolation of senescent cells using anti-DPP4 antibodies. Moreover, antibody-dependent cell-mediated cytotoxicity (ADCC) assays revealed that the cell surface DPP4 preferentially sensitized senescent, but not dividing, fibroblasts to cytotoxicity by natural killer cells. In sum, the selective expression of DPP4 on the surface of senescent cells enables their preferential elimination.

HuR and GRSF1 modulate the nuclear export and mitochondrial localization of the lncRNA RMRP.

Some mitochondrial long noncoding RNAs (lncRNAs) are encoded by nuclear DNA, but the mechanisms that mediate their transport to mitochondria are poorly characterized. Using affinity RNA pull-down followed by mass spectrometry analysis, we found two RNA-binding proteins (RBPs), HuR (human antigen R) and GRSF1 (G-rich RNA sequence-binding factor 1), that associated with the nuclear DNA-encoded lncRNA RMRP and mobilized it to mitochondria. In cultured human cells, HuR bound RMRP in the nucleus and mediated its CRM1 (chromosome region maintenance 1)-dependent export to the cytosol. After RMRP was imported into mitochondria, GRSF1 bound RMRP and increased its abundance in the matrix. Loss of GRSF1 lowered the mitochondrial levels of RMRP, in turn suppressing oxygen consumption rates and modestly reducing mitochondrial DNA replication priming. Our findings indicate that RBPs HuR and GRSF1 govern the cytoplasmic and mitochondrial localization of the lncRNA RMRP, which is encoded by nuclear DNA but has key functions in mitochondria.

circSamd4 represses myogenic transcriptional activity of PUR proteins.

By interacting with proteins and nucleic acids, the vast family of mammalian circRNAs is proposed to influence many biological processes. Here, RNA sequencing analysis of circRNAs differentially expressed during myogenesis revealed that circSamd4 expression increased robustly in mouse C2C12 myoblasts differentiating into myotubes. Moreover, silencing circSamd4, which is conserved between human and mouse, delayed myogenesis and lowered the expression of myogenic markers in cultured myoblasts from both species. Affinity pulldown followed by mass spectrometry revealed that circSamd4 associated with PURA and PURB, two repressors of myogenesis that inhibit transcription of the myosin heavy chain (MHC) protein family. Supporting the hypothesis that circSamd4 might complex with PUR proteins and thereby prevent their interaction with DNA, silencing circSamd4 enhanced the association of PUR proteins with the Mhc promoter, while overexpressing circSamd4 interfered with the binding of PUR proteins to the Mhc promoter. These effects were abrogated when using a mutant circSamd4 lacking the PUR binding site. Our results indicate that the association of PUR proteins with circSamd4 enhances myogenesis by contributing to the derepression of MHC transcription.

Transcriptome signature of cellular senescence.

Cellular senescence, an integral component of aging and cancer, arises in response to diverse triggers, including telomere attrition, macromolecular damage and signaling from activated oncogenes. At present, senescent cells are identified by the combined presence of multiple traits, such as senescence-associated protein expression and secretion, DNA damage and ß-galactosidase activity; unfortunately, these traits are neither exclusively nor universally present in senescent cells. To identify robust shared markers of senescence, we have performed RNA-sequencing analysis across eight diverse models of senescence triggered in human diploid fibroblasts (WI-38, IMR-90) and endothelial cells (HUVEC, HAEC) by replicative exhaustion, exposure to ionizing radiation or doxorubicin, and expression of the oncogene HRASG12V. The intersection of the altered transcriptomes revealed 50 RNAs consistently elevated and 18 RNAs consistently reduced across all senescence models, including many protein-coding mRNAs and some non-coding RNAs. We propose that these shared transcriptome profiles will enable the identification of senescent cells in vivo, the investigation of their roles in aging and malignancy and the development of strategies to target senescent cells therapeutically.

Loss of RNA-binding protein GRSF1 activates mTOR to elicit a proinflammatory transcriptional program.

The RNA-binding protein GRSF1 (G-rich RNA sequence-binding factor 1) critically maintains mitochondrial homeostasis. Accordingly, loss of GRSF1 impaired mitochondrial respiration and increased the levels of reactive oxygen species (ROS), triggering DNA damage, growth suppression, and a senescent phenotype characterized by elevated production and secretion of interleukin (IL)6. Here, we characterize the pathways that govern IL6 production in response to mitochondrial dysfunction in GRSF1-depleted cells. We report that loss of GRSF1 broadly altered protein expression programs, impairing the function of respiratory complexes I and IV. The rise in oxidative stress led to increased DNA damage and activation of mTOR, which in turn activated NF-κB to induce IL6 gene transcription and orchestrate a pro-inflammatory program. Collectively, our results indicate that GRSF1 helps preserve mitochondrial homeostasis, in turn preventing oxidative DNA damage and the activation of mTOR and NF-κB, and suppressing a transcriptional pro-inflammatory program leading to increased IL6 production.

Staufen1-mediated mRNA decay functions in adipogenesis.

The double-stranded RNA binding protein Staufen1 (Stau1) is involved in diverse gene expression pathways. For Stau1-mediated mRNA decay (SMD) in mammals, Stau1 binds to the 3' untranslated region of target mRNA and recruits Upf1 to elicit rapid mRNA degradation. However, the events downstream of Upf1 recruitment and the biological importance of SMD remain unclear. Here we show that SMD involves PNRC2, decapping activity, and 5'-to-3' exonucleolytic activity. In particular, Upf1 serves as an adaptor protein for the association of PNRC2 and Stau1. During adipogenesis, Stau1 and PNRC2 increase in abundance, Upf1 becomes hyperphosphorylated, and consequently SMD efficiency is enhanced. Intriguingly, downregulation of SMD components attenuates adipogenesis in a way that is rescued by downregulation of an antiadipogenic factor, Krüppel-like factor 2 (KLF2), the mRNA of which is identified as a substrate of SMD. Our data thus identify a biological role for SMD in adipogenesis.

Identification of senescence-associated circular RNAs (SAC-RNAs) reveals senescence suppressor CircPVT1.

Using RNA sequencing (RNA-Seq), we compared the expression patterns of circular RNAs in proliferating (early-passage) and senescent (late-passage) human diploid WI-38 fibroblasts. Among the differentially expressed senescence-associated circRNAs (which we termed 'SAC-RNAs'), we identified CircPVT1, generated by circularization of an exon of the PVT1 gene, as a circular RNA showing markedly reduced levels in senescent fibroblasts. Reducing CircPVT1 levels in proliferating fibroblasts triggered senescence, as determined by a rise in senescence-associated ß-galactosidase activity, higher abundance of CDKN1A/P21 and TP53, and reduced cell proliferation. Although several microRNAs were predicted to bind CircPVT1, only let-7 was found enriched after pulldown of endogenous CircPVT1, suggesting that CircPVT1 might selectively modulate let-7 activity and hence expression of let-7-regulated mRNAs. Reporter analysis revealed that CircPVT1 decreased the cellular pool of available let-7, and antagonizing endogenous let-7 triggered cell proliferation. Importantly, silencing CircPVT1 promoted cell senescence and reversed the proliferative phenotype observed after let-7 function was impaired. Consequently, the levels of several proliferative proteins that prevent senescence, such as IGF2BP1, KRAS and HMGA2, encoded by let-7 target mRNAs, were reduced by silencing CircPVT1. Our findings indicate that the SAC-RNA CircPVT1, elevated in dividing cells and reduced in senescent cells, sequesters let-7 to enable a proliferative phenotype.

Long noncoding RNAs in diseases of aging.

Aging is a process during which progressive deteriorating of cells, tissues, and organs over time lead to loss of function, disease, and death. Towards the goal of extending human health span, there is escalating interest in understanding the mechanisms that govern aging-associated pathologies. Adequate regulation of expression of coding and noncoding genes is critical for maintaining organism homeostasis and preventing disease processes. Long noncoding RNAs (lncRNAs) are increasingly recognized as key regulators of gene expression at all levels--transcriptional, post-transcriptional and post-translational. In this review, we discuss our emerging understanding of lncRNAs implicated in aging illnesses. We focus on diseases arising from age-driven impairment in energy metabolism (obesity, diabetes), the declining capacity to respond homeostatically to proliferative and damaging stimuli (cancer, immune dysfunction), and neurodegeneration. We identify the lncRNAs involved in these ailments and discuss the rising interest in lncRNAs as diagnostic and therapeutic targets to ameliorate age-associated pathologies and prolong health. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.

Identification of HuR target circular RNAs uncovers suppression of PABPN1 translation by CircPABPN1.

HuR influences gene expression programs and hence cellular phenotypes by binding to hundreds of coding and noncoding linear RNAs. However, whether HuR binds to circular RNAs (circRNAs) and impacts on their function is unknown. Here, we have identified en masse circRNAs binding HuR in human cervical carcinoma HeLa cells. One of the most prominent HuR target circRNAs was hsa_circ_0031288, renamed CircPABPN1 as it arises from the PABPN1 pre-mRNA. Further analysis revealed that HuR did not influence CircPABPN1 abundance; interestingly, however, high levels of CircPABPN1 suppressed HuR binding to PABPN1 mRNA. Evaluation of PABPN1 mRNA polysomes indicated that PABPN1 translation was modulated positively by HuR and hence negatively by CircPABPN1. We propose that the extensive binding of CircPABPN1 to HuR prevents HuR binding to PABPN1 mRNA and lowers PABPN1 translation, providing the first example of competition between a circRNA and its cognate mRNA for an RBP that affects translation.

Human proline-rich nuclear receptor coregulatory protein 2 mediates an interaction between mRNA surveillance machinery and decapping complex.

Nonsense-mediated mRNA decay (NMD) is the best-characterized mRNA surveillance mechanism by which aberrant mRNAs harboring premature termination codons are degraded before translation. However, to date, how NMD machinery recruits the general decay complex to faulty mRNAs and degrades those mRNAs remains unclear. Here we identify human proline-rich nuclear receptor coregulatory protein 2 (PNRC2) as a Upf1- and Dcp1a-interacting protein. Downregulation of PNRC2 abrogates NMD, and artificially tethering PNRC2 downstream of a normal termination codon reduces mRNA abundance. Accordingly, PNRC2 preferentially interacts with hyperphosphorylated Upf1 compared with wild-type Upf1 and triggers movement of hyperphosphorylated Upf1 into processing bodies (P bodies). Our observations suggest that PNRC2 plays an essential role in mammalian NMD, mediating the interaction between the NMD machinery and the decapping complex, so as to target the aberrant mRNA-containing RNPs into P bodies.

A new MIF4G domain-containing protein, CTIF, directs nuclear cap-binding protein CBP80/20-dependent translation.

During or right after mRNA export via the nuclear pore complex (NPC) in mammalian cells, mRNAs undergo translation mediated by nuclear cap-binding proteins 80 and 20 (CBP80/20). After CBP80/20-dependent translation, CBP80/20 is replaced by cytoplasmic cap-binding protein eIF4E, which directs steady-state translation. Nonsense-mediated mRNA decay (NMD), one of the best-characterized mRNA surveillance mechanisms, has been shown to occur on CBP80/20-bound mRNAs. However, despite the tight link between CBP80/20-dependent translation and NMD, the underlying molecular mechanism and cellular factors that mediate CBP80/20-dependent translation remain obscure. Here, we identify a new MIF4G domain-containing protein, CTIF (CBP80/20-dependent translation initiation factor). CTIF interacts directly with CBP80 and is part of the CBP80/20-dependent translation initiation complex. Depletion of endogenous CTIF from an in vitro translation system selectively blocks the translation of CBP80-bound mRNAs, while addition of purified CTIF restores it. Accordingly, down-regulation of endogenous CTIF abrogates NMD. Confocal microscopy shows that CTIF is localized to the perinuclear region. Our observations demonstrate the existence of CBP80/20-dependent translation and support the idea that CBP80/20-dependent translation is mechanistically different from steady-state translation through identification of a specific cellular protein, CTIF.

Rapid degradation of replication-dependent histone mRNAs largely occurs on mRNAs bound by nuclear cap-binding proteins 80 and 20.

The translation of mammalian messenger RNAs (mRNAs) can be driven by either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF)4E. Although CBP80/20-dependent translation (CT) is known to be coupled to an mRNA surveillance mechanism termed nonsense-mediated mRNA decay (NMD), its molecular mechanism and biological role remain obscure. Here, using a yeast two-hybrid screening system, we identify a stem-loop binding protein (SLBP) that binds to a stem-loop structure at the 3'-end of the replication-dependent histone mRNA as a CT initiation factor (CTIF)-interacting protein. SLBP preferentially associates with the CT complex of histone mRNAs, but not with the eIF4E-depedent translation (ET) complex. Several lines of evidence indicate that rapid degradation of histone mRNA on the inhibition of DNA replication largely takes place during CT and not ET, which has been previously unappreciated. Furthermore, the ratio of CBP80/20-bound histone mRNA to eIF4E-bound histone mRNA is larger than the ratio of CBP80/20-bound polyadenylated ß-actin or eEF2 mRNA to eIF4E-bound polyadenylated ß-actin or eEF2 mRNA, respectively. The collective findings suggest that mRNAs harboring a different 3'-end use a different mechanism of translation initiation, expanding the repertoire of CT as a step for determining the fate of histone mRNAs.

Predictors of Depression in Elderly According to Gender during COVID-19: Using the Data of 2020 Community Health Survey.

BACKGROUND: This study aimed to examine factors influencing depression according to gender in people during COVID-19. METHODS: This study was conducted on 61,147 elderly individuals over the age of 65 who participated in the 2020 Community Health Survey (CHS). Data analysis was conducted using SAS 9.4. RESULTS: Elderly females had a higher perceived fear of COVID-19 than males. The common factors affecting depression in elderly individuals were age, monthly income, economic activity, stress, subjective health status, and social support. Among elderly women, changes in residential areas and daily life due to COVID-19 were identified as factors affecting depression. CONCLUSIONS: Therefore, during COVID-19, it was necessary to provide customized depression relief programs for the elderly, and it was necessary to find ways for them to positively perceive their health status and increase healthcare efficacy. In the future, it is necessary to pay attention to elderly women living in rural areas and make efforts to ensure that their daily lives are not interrupted by infectious diseases.

Anti-Melanogenic Effects of Cnidium monnieri Extract via p38 Signaling-Mediated Proteasomal Degradation of Tyrosinase.

Cnidium monnieri fructus is widely used in traditional Oriental medicine for treating female genital disorders, male impotence, frigidity, and skin-related conditions in East Asia. However, the role of C. monnieri fructus extract (CMFE) in melanin synthesis is not well elucidated. This study aimed to investigate the anti-melanogenesis effect and mechanism of action of CMFE in α-MSH-stimulated B16F10 cells. Intracellular melanin content and tyrosinase activity were measured in α-MSH-stimulated B16F10 cells treated with various concentrations of CMFE (0.5-5 µg/mL). mRNA and protein levels of tyrosinase and MITF were evaluated using qRT-PCR and ting. CMFE's effect on the proteasomal degradation of tyrosinase was confirmed using a proteasomal degradation inhibitor, MG132. CMFE treatment activated p38, a protein associated with proteasomal degradation. Treatment with CMFE at up to 5 µg/mL showed no significant cytotoxicity. CMFE significantly reduced α-MSH-stimulated melanin production (43.29 ± 3.55% decrease, p < 0.05) and cellular tyrosinase activity (31.14 ± 3.15% decrease, p < 0.05). Although mRNA levels of MITF and tyrosinase increased, CMFE suppressed tyrosinase protein levels. The suppressive effect of CMFE on tyrosinase protein was blocked by MG132. CMFE inhibited melanogenesis by promoting the proteasome degradation of tyrosinase through p38 activation. These findings suggest that CMFE has the potential to be a natural whitening agent for inhibiting melanogenesis.

Utilization of nicking properties of CRISPR-Cas12a effector for genome editing.

The CRISPR-Cas nickase system for genome editing has attracted considerable attention owing to its safety, efficiency, and versatility. Although alternative effectors to Cas9 have the potential to expand the scope of genome editing, their application has not been optimized. Herein, we used an enhanced CRISPR-Cas12a nickase system to induce mutations by targeting genes in a human-derived cell line. The optimized CRISPR-Cas12a nickase system effectively introduced mutations into target genes under a specific directionality and distance between nickases. In particular, the single-mode Cas12a nickase system can induce the target-specific mutations with less DNA double-strand breaks. By inducing mutations in the Thymine-rich target genes in single- or dual-mode, Cas12a nickase compensates the limitations of Cas9 nickase and is expected to contribute to the development of future genome editing technologies.

Epidemiological investigation of a food-borne outbreak in a kindergarten, Jeju Province, Korea.

OBJECTIVES: On Monday, September 6, 2021, at a kindergarten in Jeju Province, a large number of children vomited and developed food poisoning symptoms, and this necessitated an epidemiological investigation. METHODS: The team surveyed symptoms and food intake history of kindergarten children, teachers, and workers who ate lunch between September 2 (Thursday) and September 6 (Monday), excluding weekends. In addition to rectal swabs, environmental samples from preserved foods, cooking utensils, drinking water, and refrigerator handles were collected. Pulsed field gel electrophoresis (PFGE) for genetic fingerprint analysis was also performed. RESULTS: There were 19 cases among 176 subjects, which indicated an attack rate of 10.8%. The epidemic curve showed a unimodal shape, and the average incubation period was 2.6 hours. While no food was statistically significant in food intake history, the analysis of 35 rectal smear samples detected Bacillus cereus in 7 children, 4 teachers, and 1 cooking staff. Enterotoxins were also detected in 12 samples. Out of 38 environmental samples, B. cereus and enterotoxins were detected in the morning snack cereal, lunch bean sprouts, and afternoon snack steamed potatoes on Monday, September 6th. The result of the PFGE test on 10 isolates of B. cereus showed that there was no genetic homology. CONCLUSIONS: Our results indicated that this outbreak was simultaneously caused by various strains of B. cereus from the environment.

RNA-binding proteins in cellular senescence.

Cellular senescence is a state of irreversible cell cycle arrest that is triggered and controlled by various external and/or internal factors. Among them, the regulation of senescence-associated genes is an important molecular event that plays a role in senescence. The regulation of gene expression can be achieved by various types of modulating mechanisms, and RNA-binding proteins (RBPs) are commonly known as critical regulators targeting a global range of transcripts. RBPs bind to RNA-binding motifs of the target transcripts and are involved in post-transcriptional processes such as RNA transport, stabilization, splicing, and decay. These RBPs may also play critical roles in cellular senescence by regulating the expression of senescence-associated genes. The biological functions of RBPs in controlling cellular senescence are being actively studied. Herein, we summarized the RBPs that influence cellular senescence, particularly by regulating processes such as the senescence-associated secretory phenotype, cell cycle, and mitochondrial function.

The upstream open reading frame of cyclin-dependent kinase inhibitor 1A mRNA negatively regulates translation of the downstream main open reading frame.

The first round of translation occurs on mRNAs bound by nuclear cap-binding complex (CBC), which is composed of nuclear cap-binding protein 80 and 20 (CBP80/20). During this round of translation, aberrant mRNAs are recognized and downregulated in abundance by nonsense-mediated mRNA decay (NMD), which is one of the mRNA quality control mechanisms. Here, our microarray analysis reveals that the level of cyclin-dependent kinase inhibitor 1A (CDKN1A; also known as Waf1/p21) mRNAs increases in cells depleted of cellular NMD factors. Intriguingly, CDKN1A mRNA contains an upstream open reading frame (uORF), which is a NMD-inducing feature. Using chimeric reporter constructs, we find that the uORF of CDKN1A mRNA negatively modulates translation of the main downstream ORF. These findings provide biological insights into the possible role of NMD in diverse biological pathways mediated by CDKN1A.