Increased PTCHD4 expression via m6A modification of PTCHD4 mRNA promotes senescent cell survival.

RNA modifications, including N6-methyladenosine (m6A), critically modulate protein expression programs in a range of cellular processes. Although the transcriptomes of cells undergoing senescence are strongly regulated, the landscape and impact of m6A modifications during senescence are poorly understood. Here, we report a robust m6A modification of PTCHD4 mRNA, encoding Patched Domain-Containing Protein 4, in senescent cells. The METTL3/METTL14 complex was found to incorporate the m6A modification on PTCHD4 mRNA; addition of m6A rendered PTCHD4 mRNA more stable and increased PTCHD4 production. MeRIP RT-qPCR and eCLIP analyses were used to map this m6A modification to the last exon of PTCHD4 mRNA. Further investigation identified IGF2BP1, but not other m6A readers, as responsible for the stabilization and increased abundance of m6A-modified PTCHD4 mRNA. Silencing PTCHD4, a transmembrane protein, enhanced growth arrest and DNA damage in pre-senescent cells and sensitized them to senolysis and apoptosis. Our results indicate that m6A modification of PTCHD4 mRNA increases the production of PTCHD4, a protein associated with senescent cell survival, supporting the notion that regulating m6A modification on specific mRNAs could be exploited to eliminate senescent cells for therapeutic benefit.

Increased ANKRD1 Levels in Early Senescence Mediated by RBMS1-Elicited ANKRD1 mRNA Stabilization.

Cellular senescence is a dynamic biological process triggered by sublethal cell damage and driven by specific changes in gene expression programs. We recently identified ANKRD1 (ankyrin repeat domain 1) as a protein strongly elevated after triggering senescence in fibroblasts. Here, we set out to investigate the mechanisms driving the elevated production of ANKRD1 in the early stages of senescence. Our results indicated that the rise in ANKRD1 levels after triggering senescence using etoposide (Eto) was the result of moderate increases in transcription and translation, and robust mRNA stabilization. Antisense oligomer (ASO) pulldown followed by mass spectrometry revealed a specific interaction of the RNA-binding protein RBMS1 with ANKRD1 mRNA that was confirmed by ribonucleoprotein immunoprecipitation analysis. RBMS1 abundance decreased in the nucleus and increased in the cytoplasm during Eto-induced senescence; in agreement with the hypothesis that RBMS1 may participate in post-transcriptional stabilization of ANKRD1 mRNA, silencing RBMS1 reduced, while overexpressing RBMS1 enhanced ANKRD1 mRNA half-life after Eto treatment. A segment proximal to the ANKRD1 coding region was identified as binding RBMS1 and conferring RBMS1-dependent increased expression of a heterologous reporter. We propose that RBMS1 increases expression of ANKRD1 during the early stages of senescence by stabilizing ANKRD1 mRNA.

Characterization of age-associated gene expression changes in mouse sweat glands.

Evaporation of sweat on the skin surface is the major mechanism for dissipating heat in humans. The secretory capacity of sweat glands (SWGs) declines during aging, leading to heat intolerance in the elderly, but the mechanisms responsible for this decline are poorly understood. We investigated the molecular changes accompanying SWG aging in mice, where sweat tests confirmed a significant reduction of active SWGs in old mice relative to young mice. We first identified SWG-enriched mRNAs by comparing the skin transcriptome of Eda mutant Tabby male mice, which lack SWGs, with that of wild-type control mice by RNA-sequencing analysis. This comparison revealed 171 mRNAs enriched in SWGs, including 47 mRNAs encoding 'core secretory' proteins such as transcription factors, ion channels, ion transporters, and trans-synaptic signaling proteins. Among these, 28 SWG-enriched mRNAs showed significantly altered abundance in the aged male footpad skin, and 11 of them, including Foxa1, Best2, Chrm3, and Foxc1 mRNAs, were found in the 'core secretory' category. Consistent with the changes in mRNA expression levels, immunohistology revealed that higher numbers of secretory cells from old SWGs express the transcription factor FOXC1, the protein product of Foxc1 mRNA. In sum, our study identified mRNAs enriched in SWGs, including those that encode core secretory proteins, and altered abundance of these mRNAs and proteins with aging in mouse SWGs.

Stoichiometry of long noncoding RNA interactions with other RNAs: Insights from OIP5-AS1.

Long noncoding (lnc)RNAs modulate gene expression programs in a range of developmental processes in different organs. In skeletal muscle, lncRNAs have been implicated in myogenesis, the process whereby muscle precursor cells form muscle fibers during embryonic development and regenerate muscle fibers in the adult. Here, we discuss OIP5-AS1, a lncRNA that is highly expressed in skeletal muscle and is capable of coordinating protein expression programs during myogenesis. Given that several myogenic functions of OIP5-AS1 involve interactions with MEF2C mRNA and with the microRNA miR-7, it was critical to carefully evaluate the precise levels of OIP5-AS1 during myogenesis. We discuss the approaches used to examine lncRNA copy number using OIP5-AS1 as an example, focusing on quantification by quantitative PCR analysis with reference to nucleic acids of known abundance, by droplet digital (dd)PCR measurement, and by microscopic visualization of individual lncRNAs in cells. We discuss considerations of RNA stoichiometry in light of developmental processes in which lncRNAs are implicated. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

Survey of organ-derived small extracellular vesicles and particles (sEVPs) to identify selective protein markers in mouse serum.

Extracellular vesicles and particles (EVPs) are secreted by organs across the body into different circulatory systems, including the bloodstream, and reflect pathophysiologic conditions of the organ. However, the heterogeneity of EVPs in the blood makes it challenging to determine their organ of origin. We hypothesized that small (s)EVPs (<100 nm in diameter) in the bloodstream carry distinctive protein signatures associated with each originating organ, and we investigated this possibility by studying the proteomes of sEVPs produced by six major organs (brain, liver, lung, heart, kidney, fat). We found that each organ contained distinctive sEVP proteins: 68 proteins were preferentially found in brain sEVPs, 194 in liver, 39 in lung, 15 in heart, 29 in kidney, and 33 in fat. Furthermore, we isolated sEVPs from blood and validated the presence of sEVP proteins associated with the brain (DPP6, SYT1, DNM1L), liver (FABPL, ARG1, ASGR1/2), lung (SFPTA1), heart (CPT1B), kidney (SLC31), and fat (GDN). We further discovered altered levels of these proteins in serum sEVPs prepared from old mice compared to young mice. In sum, we have cataloged sEVP proteins that can serve as potential biomarkers for organ identification in serum and show differential expression with age.

The YAP-TEAD complex promotes senescent cell survival by lowering endoplasmic reticulum stress.

Sublethal cell damage can trigger senescence, a complex adaptive program characterized by growth arrest, resistance to apoptosis and a senescence-associated secretory phenotype (SASP). Here, a whole-genome CRISPR knockout screen revealed that proteins in the YAP-TEAD pathway influenced senescent cell viability. Accordingly, treating senescent cells with a drug that inhibited this pathway, verteporfin (VPF), selectively triggered apoptotic cell death largely by derepressing DDIT4, which in turn inhibited mTOR. Reducing mTOR function in senescent cells diminished endoplasmic reticulum (ER) biogenesis, triggering ER stress and apoptosis due to high demands on ER function by the SASP. Importantly, VPF treatment decreased the numbers of senescent cells in the organs of old mice and mice exhibiting doxorubicin-induced senescence. Moreover, VPF treatment reduced immune cell infiltration and pro-fibrotic transforming growth factor-ß signaling in aging mouse lungs, improving tissue homeostasis. We present an alternative senolytic strategy that eliminates senescent cells by hindering ER activity required for SASP production.

Transcriptomes of human primary skin fibroblasts of healthy individuals reveal age-associated mRNAs and long noncoding RNAs.

Changes in the transcriptomes of human tissues with advancing age are poorly cataloged. Here, we sought to identify the coding and long noncoding RNAs present in cultured primary skin fibroblasts collected from 82 healthy individuals across a wide age spectrum (22-89 years old) who participated in the GESTALT (Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing) study of the National Institute on Aging, NIH. Using high-throughput RNA sequencing and a linear regression model, we identified 1437 coding RNAs (mRNAs) and 1177 linear and circular long noncoding (lncRNAs) that were differentially abundant as a function of age. Gene set enrichment analysis (GSEA) revealed select transcription factors implicated in coordinating the transcription of subsets of differentially abundant mRNAs, while long noncoding RNA enrichment analysis (LncSEA) identified RNA-binding proteins predicted to participate in the age-associated lncRNA profiles. In summary, we report age-associated changes in the global transcriptome, coding and noncoding, from healthy human skin fibroblasts and propose that these transcripts may serve as biomarkers and therapeutic targets in aging skin.

Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest.

Senescent cells release a variety of cytokines, proteases, and growth factors collectively known as the senescence-associated secretory phenotype (SASP). Sustained SASP contributes to a pattern of chronic inflammation associated with aging and implicated in many age-related diseases. Here, we investigated the expression and function of the immunomodulatory cytokine BAFF (B-cell activating factor; encoded by the TNFSF13B gene), a SASP protein, in multiple senescence models. We first characterized BAFF production across different senescence paradigms, including senescent human diploid fibroblasts (WI-38, IMR-90) and monocytic leukemia cells (THP-1), and tissues of mice induced to undergo senescence. We then identified IRF1 (interferon regulatory factor 1) as a transcription factor required for promoting TNFSF13B mRNA transcription in senescence. We discovered that suppressing BAFF production decreased the senescent phenotype of both fibroblasts and monocyte-like cells, reducing IL6 secretion and SA-ß-Gal staining. Importantly, however, the influence of BAFF on the senescence program was cell type-specific: in monocytes, BAFF promoted the early activation of NF-κB and general SASP secretion, while in fibroblasts, BAFF contributed to the production and function of TP53 (p53). We propose that BAFF is elevated across senescence models and is a potential target for senotherapy.

Single-cell transcriptomic analysis uncovers diverse and dynamic senescent cell populations.

Senescence is a state of enduring growth arrest triggered by sublethal cell damage. Given that senescent cells actively secrete proinflammatory and matrix-remodeling proteins, their accumulation in tissues of older persons has been linked to many diseases of aging. Despite intense interest in identifying robust markers of senescence, the highly heterogeneous and dynamic nature of the senescent phenotype has made this task difficult. Here, we set out to comprehensively analyze the senescent transcriptome of human diploid fibroblasts at the individual-cell scale by performing single-cell RNA-sequencing analysis through two approaches. First, we characterized the different cell states in cultures undergoing senescence triggered by different stresses, and found distinct cell subpopulations that expressed mRNAs encoding proteins with roles in growth arrest, survival, and the secretory phenotype. Second, we characterized the dynamic changes in the transcriptomes of cells as they developed etoposide-induced senescence; by tracking cell transitions across this process, we found two different senescence programs that developed divergently, one in which cells expressed traditional senescence markers such as p16 (CDKN2A) mRNA, and another in which cells expressed long noncoding RNAs and splicing was dysregulated. Finally, we obtained evidence that the proliferation status at the time of senescence initiation affected the path of senescence, as determined based on the expressed RNAs. We propose that a deeper understanding of the transcriptomes during the progression of different senescent cell phenotypes will help develop more effective interventions directed at this detrimental cell population.

DPP4 inhibition impairs senohemostasis to improve plaque stability in atherosclerotic mice.

Senescent vascular smooth muscle cells (VSMCs) accumulate in the vasculature with age and tissue damage and secrete factors that promote atherosclerotic plaque vulnerability and disease. Here, we report increased levels and activity of dipeptidyl peptidase 4 (DPP4), a serine protease, in senescent VSMCs. Analysis of the conditioned media from senescent VSMCs revealed a unique senescence-associated secretory phenotype (SASP) signature comprising many complement and coagulation factors; silencing or inhibiting DPP4 reduced these factors and increased cell death. Serum samples from persons with high risk for cardiovascular disease contained high levels of DPP4-regulated complement and coagulation factors. Importantly, DPP4 inhibition reduced senescent cell burden and coagulation and improved plaque stability, while single-cell resolution of senescent VSMCs reflected the senomorphic and senolytic effects of DPP4 inhibition in murine atherosclerosis. We propose that DPP4-regulated factors could be exploited therapeutically to reduce senescent cell function, reverse senohemostasis, and improve vascular disease.

Improved Macrophage Enrichment from Mouse Skeletal Muscle.

Macrophages are a heterogeneous class of innate immune cells that offer a primary line of defense to the body by phagocytizing pathogens, digesting them, and presenting the antigens to T and B cells to initiate adaptive immunity. Through specialized pro-inflammatory or anti-inflammatory activities, macrophages also directly contribute to the clearance of infections and the repair of tissue injury. Macrophages are distributed throughout the body and largely carry out tissue-specific functions. In skeletal muscle, macrophages regulate tissue repair and regeneration; however, the characteristics of these macrophages are not yet fully understood, and their involvement in skeletal muscle aging remains to be elucidated. To investigate these functions, it is critical to efficiently isolate macrophages from skeletal muscle with sufficient purity and yield for various downstream analyses. However, methods to prepare enriched skeletal muscle macrophages are scarce. Here, we describe in detail an optimized method to isolate skeletal muscle macrophages from mice. This method has allowed the isolation of CD45 + /CD11b + macrophage-enriched cells from young and old mice, which can be further used for flow cytometric analysis, fluorescence-activated cell sorting (FACS), and single-cell RNA sequencing. This protocol was validated in: eLife (2022), DOI: 10.7554/eLife.77974.

Enhanced myogenesis through lncFAM-mediated recruitment of HNRNPL to the MYBPC2 promoter.

The mammalian transcriptome comprises a vast family of long noncoding (lnc)RNAs implicated in physiologic processes such as myogenesis, through which muscle forms during embryonic development and regenerates in the adult. However, the specific molecular mechanisms by which lncRNAs regulate human myogenesis are poorly understood. Here, we identified a novel muscle-specific lncRNA, lncFAM71E1-2:2 (lncFAM), which increased robustly during early human myogenesis. Overexpression of lncFAM promoted differentiation of human myoblasts into myotubes, while silencing lncFAM suppressed this process. As lncFAM resides in the nucleus, chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS) analysis was employed to identify the molecular mechanisms whereby it might promote myogenesis. Analysis of lncFAM-interacting proteins revealed that lncFAM recruited the RNA-binding protein HNRNPL to the promoter of MYBPC2, in turn increasing MYBPC2 mRNA transcription and enhancing production of the myogenic protein MYBPC2. These results highlight a mechanism whereby a novel ribonucleoprotein complex, lncFAM-HNRNPL, elevates MYBPC2 expression transcriptionally to promote myogenesis.

Transcriptomic analysis of human ALS skeletal muscle reveals a disease-specific pattern of dysregulated circRNAs.

Circular RNAs are abundant, covalently closed transcripts that arise in cells through back-splicing and display distinct expression patterns across cells and developmental stages. While their functions are largely unknown, their intrinsic stability has made them valuable biomarkers in many diseases. Here, we set out to examine circRNA patterns in amyotrophic lateral sclerosis (ALS). By RNA-sequencing analysis, we first identified circRNAs and linear RNAs that were differentially abundant in skeletal muscle biopsies from ALS compared to normal individuals. By RT-qPCR analysis, we confirmed that 8 circRNAs were significantly elevated and 10 were significantly reduced in ALS, while the linear mRNA counterparts, arising from shared precursor RNAs, generally did not change. Several of these circRNAs were also differentially abundant in motor neurons derived from human induced pluripotent stem cells (iPSCs) bearing ALS mutations, and across different disease stages in skeletal muscle from a mouse model of ALS (SOD1G93A). Interestingly, a subset of the circRNAs significantly elevated in ALS muscle biopsies were significantly reduced in the spinal cord samples from ALS patients and ALS (SOD1G93A) mice. In sum, we have identified differentially abundant circRNAs in ALS-relevant tissues (muscle and spinal cord) that could inform about neuromuscular molecular programs in ALS and guide the development of therapies.

Context-Dependent Function of Long Noncoding RNA PURPL in Transcriptome Regulation during p53 Activation.

PURPL is a p53-induced lncRNA that suppresses basal p53 levels. Here, we investigated PURPL upon p53 activation in liver cancer cells, where it is expressed at significantly higher levels than other cell types. Using isoform sequencing, we discovered novel PURPL transcripts that have a retained intron and/or previously unannotated exons. To determine PURPL function upon p53 activation, we performed transcriptome sequencing (RNA-Seq) after depleting PURPL using CRISPR interference (CRISPRi), followed by Nutlin treatment to induce p53. Strikingly, although loss of PURPL in untreated cells altered the expression of only 7 genes, loss of PURPL resulted in altered expression of ~800 genes upon p53 activation, revealing a context-dependent function of PURPL. Pathway analysis suggested that PURPL is important for fine-tuning the expression of specific genes required for mitosis. Consistent with these results, we observed a significant decrease in the percentage of mitotic cells upon PURPL depletion. Collectively, these data identify novel transcripts from the PURPL locus and suggest that PURPL delicately moderates the expression of mitotic genes in the context of p53 activation to control cell cycle arrest.

A BDNF-TrkB autocrine loop enhances senescent cell viability.

Cellular senescence is characterized by cell cycle arrest, resistance to apoptosis, and a senescence-associated secretory phenotype (SASP) whereby cells secrete pro-inflammatory and tissue-remodeling factors. Given that the SASP exacerbates age-associated pathologies, some aging interventions aim at selectively eliminating senescent cells. In this study, a drug library screen uncovered TrkB (NTRK2) inhibitors capable of triggering apoptosis of several senescent, but not proliferating, human cells. Senescent cells expressed high levels of TrkB, which supported senescent cell viability, and secreted the TrkB ligand BDNF. The reduced viability of senescent cells after ablating BDNF signaling suggested an autocrine function for TrkB and BDNF, which activated ERK5 and elevated BCL2L2 levels, favoring senescent cell survival. Treatment with TrkB inhibitors reduced the accumulation of senescent cells in aged mouse organs. We propose that the activation of TrkB by SASP factor BDNF promotes cell survival and could be exploited therapeutically to reduce the senescent-cell burden.

Single-cell analysis of skeletal muscle macrophages reveals age-associated functional subpopulations.

Tissue-resident macrophages represent a group of highly responsive innate immune cells that acquire diverse functions by polarizing toward distinct subpopulations. The subpopulations of macrophages that reside in skeletal muscle (SKM) and their changes during aging are poorly characterized. By single-cell transcriptomic analysis with unsupervised clustering, we found 11 distinct macrophage clusters in male mouse SKM with enriched gene expression programs linked to reparative, proinflammatory, phagocytic, proliferative, and senescence-associated functions. Using a complementary classification, membrane markers LYVE1 and MHCII identified four macrophage subgroups: LYVE1-/MHCIIhi (M1-like, classically activated), LYVE1+/MHCIIlo (M2-like, alternatively activated), and two new subgroups, LYVE1+/MHCIIhi and LYVE1-/MHCIIlo. Notably, one new subgroup, LYVE1+/MHCIIhi, had traits of both M2 and M1 macrophages, while the other new subgroup, LYVE1-/MHCIIlo, displayed strong phagocytic capacity. Flow cytometric analysis validated the presence of the four macrophage subgroups in SKM and found that LYVE1- macrophages were more abundant than LYVE1+ macrophages in old SKM. A striking increase in proinflammatory markers (S100a8 and S100a9 mRNAs) and senescence-related markers (Gpnmb and Spp1 mRNAs) was evident in macrophage clusters from older mice. In sum, we have identified dynamically polarized SKM macrophages and propose that specific macrophage subpopulations contribute to the proinflammatory and senescent traits of old SKM.

LncRNA OIP5-AS1-directed miR-7 degradation promotes MYMX production during human myogenesis.

Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) modulate gene expression programs in physiology and disease. Here, we report a noncoding RNA regulatory network that modulates myoblast fusion into multinucleated myotubes, a process that occurs during muscle development and muscle regeneration after injury. In early stages of human myogenesis, the levels of lncRNA OIP5-AS1 increased, while the levels of miR-7 decreased. Moreover, OIP5-AS1 bound and induced miR-7 decay via target RNA-directed miRNA decay; accordingly, loss of OIP5-AS1 attenuated, while antagonizing miR-7 accelerated, myotube formation. We found that the OIP5-AS1-mediated miR-7 degradation promoted myoblast fusion, as it derepressed the miR-7 target MYMX mRNA, which encodes the fusogenic protein myomixer (MYMX). Remarkably, an oligonucleotide site blocker interfered with the OIP5-AS1-directed miR-7 degradation, allowing miR-7 to accumulate, lowering MYMX production and suppressing myotube formation. These results highlight a mechanism whereby lncRNA OIP5-AS1-mediated miR-7 decay promotes myotube formation by stimulating a myogenic fusion program.

Integrated lncRNA function upon genomic and epigenomic regulation.

Although some long noncoding (lnc)RNAs are known since the 1950s, the past 25 years have uncovered myriad lncRNAs with diverse sequences, structures, and functions. The advent of high-throughput and sensitive technologies has further uncovered the vast heterogeneity of lncRNA-interacting molecules and patterns of expressed lncRNAs. We propose a unifying functional theme for the expansive family of lncRNAs. At an elementary level, the genomic program of gene expression is elicited via canonical transcription and post-transcriptional mRNA assembly, turnover, and translation. Building upon this regulation, an epigenomic program refines the basic genomic control by modifying chromatin architecture as well as DNA and RNA chemistry. Superimposed over the genomic and epigenomic programs, lncRNAs create an additional regulatory dimension: by interacting with the proteins and nucleic acids that regulate gene expression in the nucleus and cytoplasm, lncRNAs help establish robust, nimble, and specific transcriptional and post-transcriptional control. We describe our present understanding of lncRNA-coordinated control of protein programs and cell fate and discuss challenges and opportunities as we embark on the next 25 years of lncRNA discovery.