Complex higher-order RNA structures play critical roles in all facets of gene expression; however, the through-space interaction networks that define tertiary structures and govern sampling of multiple conformations are poorly understood. Here we describe single-molecule RNA structure analysis in which multiple sites of chemical modification are identified in single RNA strands by massively parallel sequencing and then analyzed for correlated and clustered interactions. The strategy thus identifies RNA interaction groups by mutational profiling (RING-MaP) and makes possible two expansive applications. First, we identify throughspace interactions, create 3D models for RNAs spanning 80-265 nucleotides, and characterize broad classes of intramolecular interactions that stabilize RNA. Second, we distinguish distinct conformations in solution ensembles and reveal previously undetected hidden states and large-scale structural reconfigurations that occur in unfolded RNAs relative to native states. RING-MaP single-molecule nucleic acid structure interrogation enables concise and facile analysis of the global architectures and multiple conformations that govern function in RNA. These functions are mediated by tiered levels of information: The simplest is the primary sequence, and the most complex is the higher-order structure that governs interactions with ligands, proteins, and other RNAs (1, 2). Many RNAs can form more than one stable structure, and these distinct conformations often have different biological activities (3, 4). Currently, the rate of describing new RNA sequences vastly exceeds abilities to examine their structures.Here we characterize through-space interactions and multiple conformations in single RNAs by melding chemical probing and massively parallel sequencing. Because massively parallel sequencing reports the sequences of single templates, each read is fundamentally a single-molecule observation (5). We first modified RNA with a reagent that is sensitive to the underlying RNA structure and then detected multiple adducts in individual RNA strands (Fig. 1). Chemical adducts were detected as sequence mutations based on their ability to induce efficient misreading of the template nucleotide by a reverse transcriptase enzyme, an approach called mutational profiling, or MaP (6). Singlemolecule probing data were used in two distinct ways: to detect correlated RNA modifications reflecting higher-order through-space interactions (Fig. 1A) and to examine multiple conformations in single in-solution ensembles (Fig. 1B). Results and DiscussionMultisite Dimethyl Sulfate Reactivity with RNA. We used dimethyl sulfate (DMS) to probe the structures of three RNAs: the Escherichia coli thiamine pyrophosphate (TPP) riboswitch (79 nt) (7), the Tetrahymena group I intron P546 domain (160 nt) (8), and the Bacillus stearothermophilus RNase P catalytic domain (265 nt) (9). RNAs were selected to illustrate distinct RNA folding features and to emphasize increasingly difficult analysis challenges. The TPP riboswitch binds the...

Salivary adenoid cystic carcinoma (SACC) is a peculiar malignant tumor, characterized by its slow but inexorable growth, with a high incidence of lung metastasis and poor prognosis. Here, we show the upregulated expression of EGFR ligand epiregulin in a subset of SACC cells correlates with lung metastasis and unfavorable outcome in patients with SACC. We found that upregulation of epiregulin in SACC cells induced epithelial-mesenchymal transition by regulating GLI1/E-cadherin. Elevated epiregulin increased the expression of pro-angiogenic factors, such as VEGFA, bFGF, and IL-8. We also show that epiregulin can be delivered via exosomes and was enriched in exosomes derived from epiregulin-overexpressing SACC cells. Furthermore, treating immunodeficient mice with these epiregulin-enriched exosomes greatly enhanced SACC metastasis to lung. These epiregulin-enriched exosomes significantly enhanced angiogenesis in the neighboring tumor microenvironment and increased vascular permeability in the pre-metastatic lung microenvironment in vivo. Therefore, epiregulin, as well as epiregulin-containing exosomes, may be a novel target for controlling SACC lung metastasis.

Objectives To evaluate the effect of stem cells  from  exfoliated  deciduous  teeth on the hyposalivation caused by Sjögren syndrome (SS) and investigate the mechanism. Methods Stem cells were injected into the tail veins of non‐obese diabetic mice, the animal model of SS. The saliva flow was measured after pilocarpine intraperitoneal injection. Apoptosis and autophagy were evaluated by TUNEL and Western blot. Lymphocyte proportions were detected by flow cytometer. Results Fluid secretion was decreased in 21‐week‐old mice. Stem cell treatment increased fluid secretion, alleviated inflammation in the submandibular glands and reduced inflammatory cytokine levels in the serum, submandibular glands and saliva. Stem cells decreased the apoptotic cell number and the expressions of ATG5 and Beclin‐1 in the submandibular glands. Stem cells have no effect on other organs. Furthermore, the infused stem cells migrated to the spleen and liver, not the submandibular gland. Stem cells directed T cells towards Treg cells and suppressed Th1 and Tfh cells in spleen lymphocytes. Conclusion Stem cells  from  exfoliated  deciduous  teeth alleviate the hyposalivation caused by SS via decreasing the inflammatory cytokines, regulating the inflammatory microenvironment and decreasing the apoptosis and autophagy. The stem cells regulated in T‐cell differentiation are involved in the immunomodulatory effects.

Lung metastasis is a major factor affecting long-term survival in adenoid cystic carcinoma patients. Here, we showed that the long noncoding RNA (lncRNA) MRPL23 antisense RNA 1 (MRPL23-AS1) was highly expressed and correlated with lung metastasis and overall survival in salivary adenoid cystic carcinoma (SACC) patients. MRPL23-AS1 positively regulated epithelial-mesenchymal transition by forming a RNA-protein complex with enhancer of zeste homolog 2 (EZH2).MRPL23-AS1 increased the binding of EZH2 and H3K27me3 on the E-cadherin promoter region. Moreover, MRPL23-AS1 levels were higher in exosomes isolated from the blood plasma of SACC patients, and exosomal MRPL23-AS1 affected pulmonary microvascular endothelial cells in an 'exosomecrine' manner.MRPL23-AS1-enriched exosomes increased microvascular permeability and facilitated the metastasis of SACC in vivo. Collectively, these findings highlight a molecular mechanism of lung metastasis in SACC. MRPL23-AS1 may represent a biomarker and target for clinical intervention to control this intractable disease.