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.

Transcriptomic and Proteomic of Gastrocnemius Muscle in Peripheral Artery Disease.

BACKGROUND: Few effective therapies exist to improve lower extremity muscle pathology and mobility loss due to peripheral artery disease (PAD), in part because mechanisms associated with functional impairment remain unclear. METHODS: To better understand mechanisms of muscle impairment in PAD, we performed in-depth transcriptomic and proteomic analyses on gastrocnemius muscle biopsies from 31 PAD participants (mean age, 69.9 years) and 29 age- and sex-matched non-PAD controls (mean age, 70.0 years) free of diabetes or limb-threatening ischemia. RESULTS: Transcriptomic and proteomic analyses suggested activation of hypoxia-compensatory mechanisms in PAD muscle, including inflammation, fibrosis, apoptosis, angiogenesis, unfolded protein response, and nerve and muscle repair. Stoichiometric proportions of mitochondrial respiratory proteins were aberrant in PAD compared to non-PAD, suggesting that respiratory proteins not in complete functional units are not removed by mitophagy, likely contributing to abnormal mitochondrial activity. Supporting this hypothesis, greater mitochondrial respiratory protein abundance was significantly associated with greater complex II and complex IV respiratory activity in non-PAD but not in PAD. Rate-limiting glycolytic enzymes, such as hexokinase and pyruvate kinase, were less abundant in muscle of people with PAD compared with non-PAD participants, suggesting diminished glucose metabolism. CONCLUSIONS: In PAD muscle, hypoxia induces accumulation of mitochondria respiratory proteins, reduced activity of rate-limiting glycolytic enzymes, and an enhanced integrated stress response that modulates protein translation. These mechanisms may serve as targets for disease modification.

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.

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.

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.

Identification of atrial-enriched lncRNA Walras linked to cardiomyocyte cytoarchitecture and atrial fibrillation.

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia in humans. Genetic and genomic analyses have recently demonstrated that the homeobox transcription factor Pitx2 plays a fundamental role regulating expression of distinct growth factors, microRNAs and ion channels leading to morphological and molecular alterations that promote the onset of AF. Here we address the plausible contribution of long non-coding (lnc)RNAs within the Pitx2>Wnt>miRNA signaling pathway. In silico analyses of annotated lncRNAs in the vicinity of the Pitx2, Wnt8 and Wnt11 chromosomal loci identified five novel lncRNAs with differential expression during cardiac development. Importantly, three of them, Walaa, Walras, and Wallrd, are evolutionarily conserved in humans and displayed preferential atrial expression during embryogenesis. In addition, Walrad displayed moderate expression during embryogenesis but was more abundant in the right atrium. Walaa, Walras and Wallrd were distinctly regulated by Pitx2, Wnt8, and Wnt11, and Wallrd was severely elevated in conditional atrium-specific Pitx2-deficient mice. Furthermore, pro-arrhythmogenic and pro-hypertrophic substrate administration to primary cardiomyocyte cell cultures consistently modulate expression of these lncRNAs, supporting distinct modulatory roles of the AF cardiovascular risk factors in the regulation of these lncRNAs. Walras affinity pulldown assays revealed its association with distinct cytoplasmic and nuclear proteins previously involved in cardiac pathophysiology, while loss-of-function assays further support a pivotal role of this lncRNA in cytoskeletal organization. We propose that lncRNAs Walaa, Walras and Wallrd, distinctly regulated by Pitx2>Wnt>miRNA signaling and pro-arrhythmogenic and pro-hypertrophic factors, are implicated in atrial arrhythmogenesis, and Walras additionally in cardiomyocyte cytoarchitecture.

Reduction of lamin B receptor levels by miR-340-5p disrupts chromatin, promotes cell senescence and enhances senolysis.

A major stress response influenced by microRNAs (miRNAs) is senescence, a state of indefinite growth arrest triggered by sublethal cell damage. Here, through bioinformatic analysis and experimental validation, we identified miR-340-5p as a novel miRNA that foments cellular senescence. miR-340-5p was highly abundant in diverse senescence models, and miR-340-5p overexpression in proliferating cells rendered them senescent. Among the target mRNAs, miR-340-5p prominently reduced the levels of LBR mRNA, encoding lamin B receptor (LBR). Loss of LBR by ectopic overexpression of miR-340-5p derepressed heterochromatin in lamina-associated domains, promoting the expression of DNA repetitive elements characteristic of senescence. Importantly, overexpressing miR-340-5p enhanced cellular sensitivity to senolytic compounds, while antagonization of miR-340-5p reduced senescent cell markers and engendered resistance to senolytic-induced cell death. We propose that miR-340-5p can be exploited for removing senescent cells to restore tissue homeostasis and mitigate damage by senescent cells in pathologies of human aging.

AUF1 ligand circPCNX reduces cell proliferation by competing with p21 mRNA to increase p21 production.

Mammalian circRNAs can influence different cellular processes by interacting with proteins and other nucleic acids. Here, we used ribonucleoprotein immunoprecipitation (RIP) analysis to identify systematically the circRNAs associated with the cancer-related protein AUF1. Among the circRNAs interacting with AUF1 in HeLa (human cervical carcinoma) cells, we focused on hsa_circ_0032434 (circPCNX), an abundant target of AUF1. Overexpression of circPCNX specifically interfered with the binding of AUF1 to p21 (CDKN1A) mRNA, thereby promoting p21 mRNA stability and elevating the production of p21, a major inhibitor of cell proliferation. Conversely, silencing circPCNX increased AUF1 binding to p21 mRNA, reducing p21 production and promoting cell division. Importantly, eliminating the AUF1-binding region of circPCNX abrogated the rise in p21 levels and rescued proliferation. Therefore, we propose that the interaction of circPCNX with AUF1 selectively prevents AUF1 binding to p21 mRNA, leading to enhanced p21 mRNA stability and p21 protein production, thereby suppressing cell growth.

Interaction of OIP5-AS1 with MEF2C mRNA promotes myogenic gene expression.

Long noncoding (lnc)RNAs potently regulate gene expression programs in physiology and disease. Here, we describe a key function for lncRNA OIP5-AS1 in myogenesis, the process whereby myoblasts differentiate into myotubes during muscle development and muscle regeneration after injury. In human myoblasts, OIP5-AS1 levels increased robustly early in myogenesis, and its loss attenuated myogenic differentiation and potently reduced the levels of the myogenic transcription factor MEF2C. This effect relied upon the partial complementarity of OIP5-AS1 with MEF2C mRNA and the presence of HuR, an RNA-binding protein (RBP) with affinity for both transcripts. Remarkably, HuR binding to MEF2C mRNA, which stabilized MEF2C mRNA and increased MEF2C abundance, was lost after OIP5-AS1 silencing, suggesting that OIP5-AS1 might serve as a scaffold to enhance HuR binding to MEF2C mRNA, in turn increasing MEF2C production. These results highlight a mechanism whereby a lncRNA promotes myogenesis by enhancing the interaction of an RBP and a myogenic mRNA.

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.

Cooperative translational control of polymorphic BAFF by NF90 and miR-15a.

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.

High-purity circular RNA isolation method (RPAD) reveals vast collection of intronic circRNAs.

High-throughput RNA sequencing methods coupled with specialized bioinformatic analyses have recently uncovered tens of thousands of unique circular (circ)RNAs, but their complete sequences, genes of origin and functions are largely unknown. Given that circRNAs lack free ends and are thus relatively stable, their association with microRNAs (miRNAs) and RNA-binding proteins (RBPs) can influence gene expression programs. While exoribonuclease treatment is widely used to degrade linear RNAs and enrich circRNAs in RNA samples, it does not efficiently eliminate all linear RNAs. Here, we describe a novel method for the isolation of highly pure circRNA populations involving RNase R treatment followed by Polyadenylation and poly(A)+ RNA Depletion (RPAD), which removes linear RNA to near completion. High-throughput sequencing of RNA prepared using RPAD from human cervical carcinoma HeLa cells and mouse C2C12 myoblasts led to two surprising discoveries: (i) many exonic circRNA (EcircRNA) isoforms share an identical backsplice sequence but have different body sizes and sequences, and (ii) thousands of novel intronic circular RNAs (IcircRNAs) are expressed in cells. In sum, isolating high-purity circRNAs using the RPAD method can enable quantitative and qualitative analyses of circRNA types and sequence composition, paving the way for the elucidation of circRNA functions.

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.

Regulation of HuR structure and function by dihydrotanshinone-I.

The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3'UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.

p(7°S6K¹) in the TORC1 pathway is essential for the differentiation of Th17 Cells, but not Th1, Th2, or Treg cells in mice.

The TORC1 pathway is necessary for ribosomal biogenesis and initiation of protein translation. Furthermore, the differentiation of Th1 and Th17 cells requires TORC1 activity. To investigate the role of the TORC1 pathway in the differentiation of Th1 and/or Th17 cells in more detail, we compared the differentiation capacity of naïve T cells from wild type and p70(S6K1) knockout mice. Expression of many of the genes associated with Th17-cell differentiation, such as IL17a, IL17f, and IL-23R, were reduced in p70(S6K1) knockout mice. In contrast, the development of Th1, Th2, and Treg cells was unaffected in the absence of p70(S6K1) . Furthermore, expression of the major transcription factor in Th17-cell differentiation, retinoic acid receptor-related orphan receptor gamma T, remained unchanged. However, the acetylation of histone 3 at the promoters of IL17a and IL17f was reduced in the absence of p70(S6K1) . In accordance with the in vitro data, the kinetics, but not the development, of EAE was affected with the loss of p70(S6K1) expression. Collectively, our findings suggested that both in vitro and in vivo differentiation of Th17 cells were positively regulated by p70(S6K1) .

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.

Mechanism of Activation-Induced Downregulation of Mitofusin 2 in Human Peripheral Blood T Cells.

Mitofusin 2 (Mfn2), a mitochondrial protein, was shown to have antiproliferative properties when overexpressed. In this article, we show that activation of resting human peripheral blood T cells caused downregulation of Mfn2 levels. This downregulation of Mfn2 was blocked by different inhibitors (mTOR inhibitor rapamycin, PI3K inhibitor LY294002, and Akt inhibitor A443654), producing cells that were arrested in the G0/G1 stage of the cell cycle. Furthermore, the activation-induced downregulation of Mfn2 preceded the entry of the cells into the cell cycle, suggesting that Mfn2 downregulation is a prerequisite for activated T cell entry into the cell cycle. Accordingly, small interfering RNA-mediated knockdown of Mfn2 resulted in increased T cell proliferation. Overexpression of constitutively active AKT resulted in the downregulation of Mfn2, which can be blocked by a proteasome inhibitor. Akt-mediated downregulation of Mfn2 was via the mTORC1 pathway because this downregulation was blocked by rapamycin, and overexpression of wild-type, but not kinase-dead mTOR, caused Mfn2 downregulation. Our data suggested that activation-induced reactive oxygen species production plays an important role in the downregulation of Mfn2. Collectively, our data suggest that the PI3K-AKT-mTOR pathway plays an important role in activation-induced downregulation of Mfn2 and subsequent proliferation of resting human T cells.