Adipose Tissue Macrophage-Derived Exosomal miRNAs Can Modulate In Vivo and In Vitro Insulin Sensitivity.

MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis.

Mitochondrial Priming by CD28.

T cell receptor (TCR) signaling without CD28 can elicit primary effector T cells, but memory T cells generated during this process are anergic, failing to respond to secondary antigen exposure. We show that, upon T cell activation, CD28 transiently promotes expression of carnitine palmitoyltransferase 1a (Cpt1a), an enzyme that facilitates mitochondrial fatty acid oxidation (FAO), before the first cell division, coinciding with mitochondrial elongation and enhanced spare respiratory capacity (SRC). microRNA-33 (miR33), a target of thioredoxin-interacting protein (TXNIP), attenuates Cpt1a expression in the absence of CD28, resulting in cells that thereafter are metabolically compromised during reactivation or periods of increased bioenergetic demand. Early CD28-dependent mitochondrial engagement is needed for T cells to remodel cristae, develop SRC, and rapidly produce cytokines upon restimulation-cardinal features of protective memory T cells. Our data show that initial CD28 signals during T cell activation prime mitochondria with latent metabolic capacity that is essential for future T cell responses.

MicroRNA-221 and -222 modulate intestinal inflammatory Th17 cell response as negative feedback regulators downstream of interleukin-23.

MicroRNAs are important regulators of immune responses. Here, we show miR-221 and miR-222 modulate the intestinal Th17 cell response. Expression of miR-221 and miR-222 was induced by proinflammatory cytokines and repressed by the cytokine TGF-ß. Molecular targets of miR-221 and miR-222 included Maf and Il23r, and loss of miR-221 and miR-222 expression shifted the transcriptomic spectrum of intestinal Th17 cells to a proinflammatory signature. Although the loss of miR-221 and miR-222 was tolerated for maintaining intestinal Th17 cell homeostasis in healthy mice, Th17 cells lacking miR-221 and miR-222 expanded more efficiently in response to IL-23. Both global and T cell-specific deletion of miR-221 and miR-222 rendered mice prone to mucosal barrier damage. Collectively, these findings demonstrate that miR-221 and miR-222 are an integral part of intestinal Th17 cell response that are induced after IL-23 stimulation to constrain the magnitude of proinflammatory response.

SPARCLE, a p53-induced lncRNA, controls apoptosis after genotoxic stress by promoting PARP-1 cleavage.

p53, master transcriptional regulator of the genotoxic stress response, controls cell-cycle arrest and apoptosis following DNA damage. Here, we identify a p53-induced lncRNA suicidal PARP-1 cleavage enhancer (SPARCLE) adjacent to miR-34b/c required for p53-mediated apoptosis. SPARCLE is a ∼770-nt, nuclear lncRNA induced 1 day after DNA damage. Despite low expression (<16 copies/cell), SPARCLE deletion increases DNA repair and reduces DNA-damage-induced apoptosis as much as p53 deficiency, while its overexpression restores apoptosis in p53-deficient cells. SPARCLE does not alter gene expression. SPARCLE binds to PARP-1 with nanomolar affinity and causes apoptosis by acting as a caspase-3 cofactor for PARP-1 cleavage, which separates PARP-1's N-terminal (NT) DNA-binding domain from its catalytic domains. NT-PARP-1 inhibits DNA repair. Expressing NT-PARP-1 in SPARCLE-deficient cells increases unrepaired DNA damage and restores apoptosis after DNA damage. Thus, SPARCLE enhances p53-induced apoptosis by promoting PARP-1 cleavage, which interferes with DNA-damage repair.

Promoter and enhancer RNAs regulate chromatin reorganization and activation of miR-10b/HOXD locus, and neoplastic transformation in glioma.

miR-10b is silenced in normal neuroglial cells of the brain but commonly activated in glioma, where it assumes an essential tumor-promoting role. We demonstrate that the entire miR-10b-hosting HOXD locus is activated in glioma via the cis-acting mechanism involving 3D chromatin reorganization and CTCF-cohesin-mediated looping. This mechanism requires two interacting lncRNAs, HOXD-AS2 and LINC01116, one associated with HOXD3/HOXD4/miR-10b promoter and another with the remote enhancer. Knockdown of either lncRNA in glioma cells alters CTCF and cohesin binding, abolishes chromatin looping, inhibits the expression of all genes within HOXD locus, and leads to glioma cell death. Conversely, in cortical astrocytes, enhancer activation is sufficient for HOXD/miR-10b locus reorganization, gene derepression, and neoplastic cell transformation. LINC01116 RNA is essential for this process. Our results demonstrate the interplay of two lncRNAs in the chromatin folding and concordant regulation of miR-10b and multiple HOXD genes normally silenced in astrocytes and triggering the neoplastic glial transformation.

microRNA-induced translational control of antiviral immunity by the cap-binding protein 4EHP.

Type I interferons (IFNs) are critical cytokines in the host defense against invading pathogens. Sustained production of IFNs, however, is detrimental to the host, as it provokes autoimmune diseases. Thus, the expression of IFNs is tightly controlled. We report that the mRNA 5' cap-binding protein 4EHP plays a key role in regulating type I IFN concomitant with controlling virus replication, both in vitro and in vivo. Mechanistically, 4EHP suppresses IFN-ß production by effecting the miR-34a-induced translational silencing of Ifnb1 mRNA. miR-34a is upregulated by both RNA virus infection and IFN-ß induction, prompting a negative feedback regulatory mechanism that represses IFN-ß expression via 4EHP. These findings demonstrate the direct involvement of 4EHP in virus-induced host response, underscoring a critical translational silencing mechanism mediated by 4EHP and miR-34a to impede sustained IFN production. This study highlights an intrinsic regulatory function for miRNA and the translation machinery in maintaining host homeostasis.

miR-200 deficiency promotes lung cancer metastasis by activating Notch signaling in cancer-associated fibroblasts.

Lung adenocarcinoma, the most prevalent lung cancer subtype, is characterized by its high propensity to metastasize. Despite the importance of metastasis in lung cancer mortality, its underlying cellular and molecular mechanisms remain largely elusive. Here, we identified miR-200 miRNAs as potent suppressors for lung adenocarcinoma metastasis. miR-200 expression is specifically repressed in mouse metastatic lung adenocarcinomas, and miR-200 decrease strongly correlates with poor patient survival. Consistently, deletion of mir-200c/141 in the KrasLSL-G12D/+ ; Trp53flox/flox lung adenocarcinoma mouse model significantly promoted metastasis, generating a desmoplastic tumor stroma highly reminiscent of metastatic human lung cancer. miR-200 deficiency in lung cancer cells promotes the proliferation and activation of adjacent cancer-associated fibroblasts (CAFs), which in turn elevates the metastatic potential of cancer cells. miR-200 regulates the functional interaction between cancer cells and CAFs, at least in part, by targeting Notch ligand Jagged1 and Jagged2 in cancer cells and inducing Notch activation in adjacent CAFs. Hence, the interaction between cancer cells and CAFs constitutes an essential mechanism to promote metastatic potential.

Regulation of Zbp1 by miR-99b-5p in microglia controls the development of schizophrenia-like symptoms in mice.

Current approaches to the treatment of schizophrenia have mainly focused on the protein-coding part of the genome; in this context, the roles of microRNAs have received less attention. In the present study, we analyze the microRNAome in the blood and postmortem brains of schizophrenia patients, showing that the expression of miR-99b-5p is downregulated in both the prefrontal cortex and blood of patients. Lowering the amount of miR-99b-5p in mice leads to both schizophrenia-like phenotypes and inflammatory processes that are linked to synaptic pruning in microglia. The microglial miR-99b-5p-supressed inflammatory response requires Z-DNA binding protein 1 (Zbp1), which we identify as a novel miR-99b-5p target. Antisense oligonucleotides against Zbp1 ameliorate the pathological effects of miR-99b-5p inhibition. Our findings indicate that a novel miR-99b-5p-Zbp1 pathway in microglia might contribute to the pathogenesis of schizophrenia.

An AGO10:miR165/6 module regulates meristem activity and xylem development in the Arabidopsis root.

The RNA-silencing effector ARGONAUTE10 influences cell fate in plant shoot and floral meristems. ARGONAUTE10 also accumulates in the root apical meristem (RAM), yet its function(s) therein remain elusive. Here, we show that ARGONAUTE10 is expressed in the root cell initials where it controls overall RAM activity and length. ARGONAUTE10 is also expressed in the stele, where post-transcriptional regulation confines it to the root tip's pro-vascular region. There, variations in ARGONAUTE10 levels modulate metaxylem-vs-protoxylem specification. Both ARGONAUTE10 functions entail its selective, high-affinity binding to mobile miR165/166 transcribed in the neighboring endodermis. ARGONAUTE10-bound miR165/166 is degraded, likely via SMALL-RNA-DEGRADING-NUCLEASES1/2, thus reducing miR165/166 ability to silence, via ARGONAUTE1, the transcripts of cell fate-influencing transcription factors. These include PHABULOSA (PHB), which controls meristem activity in the initials and xylem differentiation in the pro-vasculature. During early germination, PHB transcription increases while dynamic, spatially-restricted transcriptional and post-transcriptional mechanisms reduce and confine ARGONAUTE10 accumulation to the provascular cells surrounding the newly-forming xylem axis. Adequate miR165/166 concentrations are thereby channeled along the ARGONAUTE10-deficient yet ARGONAUTE1-proficient axis. Consequently, inversely-correlated miR165/166 and PHB gradients form preferentially along the axis despite ubiquitous PHB transcription and widespread miR165/166 delivery inside the whole vascular cylinder.

MicroRNA Clustering Assists Processing of Suboptimal MicroRNA Hairpins through the Action of the ERH Protein.

Microprocessor initiates the processing of microRNAs (miRNAs) from the hairpin regions of primary transcripts (pri-miRNAs). Pri-miRNAs often contain multiple miRNA hairpins, and this clustered arrangement can assist in the processing of otherwise defective hairpins. We find that miR-451, which derives from a hairpin with a suboptimal terminal loop and a suboptimal stem length, accumulates to 40-fold higher levels when clustered with a helper hairpin. This phenomenon tolerates changes in hairpin order, linker lengths, and the identities of the helper hairpin, the recipient hairpin, the linker-sequence, and the RNA polymerase that transcribes the hairpins. It can act reciprocally and need not occur co-transcriptionally. It requires Microprocessor recognition of the helper hairpin and linkage of the two hairpins, yet predominantly manifests after helper-hairpin processing. It also requires enhancer of rudimentary homolog (ERH), which copurifies with Microprocessor and can dimerize and interact with other proteins that can dimerize, suggesting a model in which one Microprocessor recruits another Microprocessor.