DNA Topoisomerases are essential to resolve topological problems during DNA metabolism in all species. However, the prevalence and function of RNA topoisomerases remain uncertain. Here, we show that RNA topoisomerase activity is prevalent in Type IA topoisomerases from bacteria, archaea, and eukarya. Moreover, this activity always requires the conserved Type IA core domains and the same catalytic residue used in DNA topoisomerase reaction; however, it does not absolutely require the non-conserved carboxyl-terminal domain (CTD), which is necessary for relaxation reactions of supercoiled DNA. The RNA topoisomerase activity of human Top3β differs from that of Escherichia coli topoisomerase I in that the former but not the latter requires the CTD, indicating that topoisomerases have developed distinct mechanisms during evolution to catalyze RNA topoisomerase reactions. Notably, Top3β proteins from several animals associate with polyribosomes, which are units of mRNA translation, whereas the Top3 homologs from E. coli and yeast lack the association. The Top3β-polyribosome association requires TDRD3, which directly interacts with Top3β and is present in animals but not bacteria or yeast. We propose that RNA topoisomerases arose in the early RNA world, and that they are retained through all domains of DNA-based life, where they mediate mRNA translation as part of polyribosomes in animals.

A prominent phenotype triggered by the loss of mitochondrial homeostasis is cellular senescence, characterized by cessation of growth and a senescence-associated secretory phenotype (SASP). We identified the G-rich RNA sequence-binding factor 1 (GRSF1) as a major mitochondrial protein implicated in this response. GRSF1 levels declined in senescent cells through reduced protein stability, and lowering GRSF1 abundance caused mitochondrial stress leading to elevated production of superoxide, increased DNA damage foci, and diminished cell proliferation. In addition, reducing GRSF1 increased the activity of a senescence-associated β-galactosidase (SA-β-gal) and the production and secretion of the SASP factor interleukin 6 (IL6). Together, our findings indicate that the decline in GRSF1 levels during cellular senescence contributes to impairing mitochondrial function, elevating ROS and DNA damage, suppressing growth, and implementing a pro-inflammatory program.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate diverse biological processes by controlling the pattern of expressed proteins. In mammalian cells, miRNAs partially complement their target sequences leading to mRNA degradation and/or decreased mRNA translation. Here, we have analyzed transcriptome-wide changes in miRNAs in senescent relative to early-passage WI-38 human diploid fibroblasts (HDFs). Among the miRNAs downregulated with senescence were members of the let-7 family, while upregulated miRNAs included miR-1204, miR-663 and miR-519. miR-519 was recently found to reduce tumor growth at least in part by lowering the abundance of the RNA-binding protein HuR. Overexpression of miR-519a in either WI-38 or human cervical carcinoma HeLa cells triggered senescence, as measured by monitoring beta-galactosidase activity and other senescence markers. These data suggest that miR-519 can suppress tumor growth by triggering senescence and that miR-519 elicits these actions by repressing HuR expression.

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.

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.

Senescence is a state of indefinite cell cycle arrest associated with aging, cancer, and age-related diseases. Here, using label-based mass spectrometry, ribosome profiling and nanopore direct RNA sequencing, we explore the coordinated interaction of translational and transcriptional programs of human cellular senescence. We find that translational deregulation and a corresponding maladaptive integrated stress response (ISR) is a hallmark of senescence that desensitizes senescent cells to stress. We present evidence that senescent cells maintain high levels of eIF2α phosphorylation, typical of ISR activation, but translationally repress production of the stress response transcription factor 4 (ATF4) by ineffective bypass of the inhibitory upstream open reading frames. Surprisingly, ATF4 translation remains inhibited even after acute proteotoxic and amino acid starvation stressors, resulting in a highly diminished stress response. Furthermore, absent a response, stress augments the senescence secretory phenotype, thus intensifying a proinflammatory state that exacerbates disease. Our results reveal a novel mechanism that senescent cells exploit to evade an adaptive stress response and remain viable.

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.

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-β-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.

The epithelium of the intestinal mucosa is a rapidly self-renewing tissue in the body, and defects in the renewal process occur commonly in various disorders. microRNAs (miRNAs) posttranscriptionally regulate gene expression and are implicated in many aspects of cellular physiology. Here we investigate the role of miRNA-29b (miR-29b) in the regulation of normal intestinal mucosal growth and further validate its target mRNAs. miRNA expression profiling studies reveal that growth inhibition of the small intestinal mucosa is associated with increased expression of numerous miRNAs, including miR-29b. The simple systemic delivery of locked nucleic acid-modified, anti-miR-29b-reduced endogenous miR-29b levels in the small intestinal mucosa increases cyclin-dependent kinase 2 (CDK2) expression and stimulates mucosal growth. In contrast, overexpression of the miR-29b precursor in intestinal epithelial cells represses CDK2 expression and results in growth arrest in G1 phase. miR-29b represses CDK2 translation through direct interaction with the cdk2 mRNA via its 3'-untranslated region (3'-UTR), whereas point mutation of miR-29b binding site in the cdk2 3'-UTR prevents miR-29b-induced repression of CDK2 translation. These results indicate that miR-29b inhibits intestinal mucosal growth by repressing CDK2 translation.

Topoisomerases are crucial for solving DNA topological problems, but they have not been linked to RNA metabolism. Here we show that human topoisomerase 3β (Top3β) is an RNA topoisomerase that biochemically and genetically interacts with FMRP, a protein that is deficient in fragile X syndrome and is known to regulate the translation of mRNAs that are important for neuronal function, abnormalities of which are linked to autism. Notably, the FMRP-Top3β interaction is abolished by a disease-associated mutation of FMRP, suggesting that Top3β may contribute to the pathogenesis of mental disorders. Top3β binds multiple mRNAs encoded by genes with neuronal functions linked to schizophrenia and autism. Expression of one such gene, that encoding protein tyrosine kinase 2 (ptk2, also known as focal adhesion kinase or FAK), is reduced in the neuromuscular junctions of Top3β mutant flies. Synapse formation is defective in Top3β mutant flies and mice, as well as in FMRP mutant flies and mice. Our findings suggest that Top3β acts as an RNA topoisomerase and works with FMRP to promote the expression of mRNAs that are crucial for neurodevelopment and mental health.

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.

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.

Despite exhaustively informing about steady-state mRNA abundance, DNA microarrays have been used with limited success to identify regulatory transcription factors (TFs). The main limitation of this approach is that altered mRNA stability also strongly governs the patterns of expressed genes. Here, we used nuclear run-on assays and microarrays to systematically interrogate changes in nascent transcription in cells treated with the topoisomerase inhibitor camptothecin (CPT). Analysis of the promoters of coordinately transcribed genes after CPT treatment suggested the involvement of TFs c-Myb and Rfx1. The predicted CPT-dependent associations were subsequently confirmed by chromatin immunoprecipitation assays. Importantly, after RNAi-mediated knockdown of each TF, the CPT-elicited induction of c-Myb- and/or Rfx1-regulated mRNAs was diminished and the overall cellular response was impaired. The strategies described here permit the successful identification of the TFs responsible for implementing adaptive gene expression programs in response to cellular stimulation.

Long noncoding RNAs (lncRNAs) are often associated with polysomes, indicating coding potential. However, only a handful of endogenous proteins encoded by putative lncRNAs have been identified and assigned a function. Here, we report the discovery of a putative gastrointestinal-tract-specific lncRNA (LINC00675) that is regulated by the pioneer transcription factor FOXA1 and encodes a conserved small protein of 79 amino acids which we termed FORCP (FOXA1-Regulated Conserved Small Protein). FORCP transcript is undetectable in most cell types but is abundant in well-differentiated colorectal cancer (CRC) cells where it functions to inhibit proliferation, clonogenicity, and tumorigenesis. The epitope-tagged and endogenous FORCP protein predominantly localizes to the endoplasmic reticulum (ER). In response to ER stress, FORCP depletion results in decreased apoptosis. Our findings on the initial characterization of FORCP demonstrate that FORCP is a novel, conserved small protein encoded by a mis-annotated lncRNA that regulates apoptosis and tumorigenicity in well-differentiated CRC cells.

Polymorphisms in untranslated regions (UTRs) of disease-associated mRNAs can alter protein production. We recently identified a genetic variant in the 3'UTR of the TNFSF13B gene, encoding the cytokine BAFF (B-cell-activating factor), that generates an alternative polyadenylation site yielding a shorter, more actively translated variant, BAFF-var mRNA. Accordingly, individuals bearing the TNFSF13B variant had higher circulating BAFF and elevated risk of developing autoimmune diseases. Here, we investigated the molecular mechanisms controlling the enhanced translation of BAFF-var mRNA. We identified nuclear factor 90 (NF90, also known as ILF3) as an RNA-binding protein that bound preferentially the wild-type (BAFF-WT mRNA) but not BAFF-var mRNA in human monocytic leukemia THP-1 cells. NF90 selectively suppressed BAFF translation by recruiting miR-15a to the 3'UTR of BAFF-WT mRNA. Our results uncover a paradigm whereby an autoimmunity-causing BAFF polymorphism prevents NF90-mediated recruitment of microRNAs to suppress BAFF translation, raising the levels of disease-associated BAFF.

A hallmark trait of cellular senescence is the acquisition of a senescence-associated secretory phenotype (SASP). SASP factors include cytokines and their receptors (IL-6, IL-8, osteoprotegerin, GM-CSF), chemokines and their ligands (MCP-1, HCC4), and oncogenes (Gro1 and Gro2), many of them encoded by mRNAs whose stability and translation are tightly regulated. Using two models of human fibroblast senescence (WI-38 and IDH4 cells), we report the identification of RNA-binding protein NF90 as a post-transcriptional repressor of several SASP factors. In 'young', proliferating fibroblasts, NF90 was highly abundant, associated with numerous SASP mRNAs, and inhibited their expression. By contrast, senescent cells expressed low levels of NF90, thus allowing SASP factor expression to increase. NF90 elicited these effects mainly by repressing the translation of target SASP mRNAs, since silencing NF90 did not increase the steady-state levels of SASP mRNAs but elevated key SASP factors including MCP-1, GROa, IL-6, and IL-8. Our findings indicate that NF90 contributes to maintaining low levels of SASP factors in non-senescent cells, while NF90 reduction in senescent cells allows SASP factor expression to rise.

Posttranscriptional gene regulation by RNA-binding proteins, such as HuR (elavl1), fine-tune gene expression in T cells, leading to powerful effects on immune responses. HuR can stabilize target mRNAs and/or promote translation by interacting with their 3' untranslated region adenylate and uridylate-rich elements. It was previously demonstrated that HuR facilitates Th2 cytokine expression by mRNA stabilization. However, its effects upon IL-2 homeostasis and CD4+ Th2 differentiation are not as well understood. We found that optimal translation of Il2ra (CD25) required interaction of its mRNA with HuR. Conditional HuR knockout in CD4+ T cells resulted in loss of IL-2 homeostasis and defects in JAK-STAT signaling, Th2 differentiation, and cytokine production. HuR-knockout CD4+ T cells from OVA-immunized mice also failed to proliferate in response to Ag. These results demonstrate that HuR plays a pivotal role in maintaining normal IL-2 homeostasis and initiating CD4+ Th2 differentiation.

In response to stress conditions, many mammalian mRNAs accumulate in stress granules (SGs) together with numerous RNA-binding proteins that control mRNA turnover and translation. However, the signaling cascades that modulate the presence of ribonucleoprotein (RNP) complexes in SGs are poorly understood. Here, we investigated the localization of human antigen R (HuR), an mRNA-stabilizing RNA-binding protein, in SGs following exposure to the stress agent arsenite. Unexpectedly, the mobilization of HuR to SGs was prevented through the activation of Janus kinase 3 (JAK3) by the vitamin K3 analog menadione. JAK3 phosphorylated HuR at tyrosine 200, in turn inhibiting HuR localization in SGs, reducing HuR interaction with targets SIRT1 and VHL mRNAs, and accelerating target mRNA decay. Our findings indicate that HuR is tyrosine-phosphorylated by JAK3, and link this modification to HuR subcytoplasmic localization and to the fate of HuR target mRNAs.

Expression of the tumor suppressor p16(INK4a) increases during aging and replicative senescence.

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.