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.     

The QKI-5 and QKI-6 RNA Binding Proteins Regulate the Expression of MicroRNA 7 in Glial Cells

The quaking (qkI) gene encodes 3 major alternatively spliced isoforms that contain unique sequences at their C termini dictating their cellular localization. QKI-5 is predominantly nuclear, whereas QKI-6 is distributed throughout the cell and QKI-7 is cytoplasmic. The QKI isoforms are sequence-specific RNA binding proteins expressed mainly in glial cells modulating RNA splicing, export, and stability. Herein, we identify a new role for the QKI proteins in the regulation of microRNA (miRNA) processing. We observed that small interfering RNA (siRNA)-mediated QKI depletion of U343 glioblastoma cells leads to a robust increase in miR-7 expression. The processing from primary to mature miR-7 was inhibited in the presence QKI-5 and QKI-6 but not QKI-7, suggesting that the nuclear localization plays an important role in the regulation of miR-7 expression. The primary miR-7-1 was bound by the QKI isoforms in a QKI response element (QRE)-specific manner. We observed that the pri-miR-7-1 RNA was tightly bound to Drosha in the presence of the QKI isoforms, and this association was not observed in siRNA-mediated QKI or Drosha-depleted U343 glioblastoma cells. Moreover, the presence of the QKI isoforms led to an increase presence of pri-miR-7 in nuclear foci, suggesting that pri-miR-7-1 is retained in the nucleus by the QKI isoforms. miR-7 is known to target the epidermal growth factor (EGF) receptor (EGFR) 3′ untranslated region (3′-UTR), and indeed, QKI-deficient U343 cells had reduced EGFR expression and decreased ERK activation in response to EGF. Elevated levels of miR-7 are associated with cell cycle arrest, and it was observed that QKI-deficient U343 that harbor elevated levels of miR-7 exhibited defects in cell proliferation that were partially rescued by the addition of a miR-7 inhibitor. These findings suggest that the QKI isoforms regulate glial cell function and proliferation by regulating the processing of certain miRNAs.     

miR-361-regulated prohibitin inhibits mitochondrial fission and apoptosis and protects heart from ischemia injury

Cardiovascular disease remains the leading cause of morbidity and mortality worldwide. Emerging evidences suggest that the abnormal mitochondrial fission participates in pathogenesis of cardiac diseases, including myocardial infarction (MI) and heart failure. However, the molecular components regulating mitochondrial network in the heart remain largely unidentified. Here we report that miR-361 and prohibitin 1 (PHB1) constitute an axis that regulates mitochondrial fission and apoptosis. The results show that PHB1 attenuates mitochondrial fission and apoptosis in response to hydrogen peroxide treatment in cardiomyocytes. Cardiac-specific PHB1 transgenic mice show reduced mitochondrial fission and myocardial infarction sizes after myocardial infarction surgery. MiR-361 is responsible for the dysfunction of PHB1 and suppresses the translation of PHB1. Knockdown of miR-361 reduces mitochondrial fission and apoptosis in vivo and in vitro. MiR-361 cardiac-specific transgenic mice represent elevated mitochondrial fission and myocardial infarction sizes upon myocardial ischemia injury. This study identifies a novel signaling pathway composed of miR-361 and PHB1 that regulates mitochondrial fission program and apoptosis. This discovery will shed new light on the therapy of myocardial infarction and heart failure.The heart drives the blood flow in the body and it has a large requirement of energy. Mitochondria meet the high energy demand of the heart by consistently providing large amounts of ATP through oxidative phosphorylation. Thus, mitochondrial malfunction is tightly related to cardiac diseases and contributes to cardiomyocyte injury, cardiomyopathy and heart failure. Mitochondria morphology is also associated with the function. Mitochondria constantly undergo fission and fusion. Fission leads to the formation of small round mitochondria and promotes cell apoptosis,^{1, 2, 3, 4, 5, 6, 7} whereas fusion results in mitochondria elongation and have a protective role in cardiomyocytes maintenance.⁸ The above findings strongly suggest that mitochondrial fission and fusion machinery is important for cardiac function. In addition, unveiling the mechanism of mitochondrial network regulation will provide a novel therapeutic strategy for heart failure.The mitochondrial prohibitin complex is a macromolecular structure at the inner mitochondrial membrane that is composed of prohibitin 1 (PHB1) and prohibitin 2 subunits.⁹ These two proteins comprise an evolutionary conserved and ubiquitously expressed family of membrane proteins and are implicated in several important cellular processes such as mitochondrial biogenesis and function, cell proliferation, replicative senescence, and cell death.^{10, 11} The first mammalian PHB1 was identified as a potential tumor suppressor with anti-proliferative activity.¹² Recent findings suggest that PHB1 has an important role in regulating mitochondrial morphology. Loss of PHB1 results in accumulation of fragmented mitochondria in MEFs and HeLa cells.^{13, 14} However, it is not yet clear whether PHB1 participates in the regulation of mitochondrial dynamics in cardiomyocytes.MicroRNAs (miRNAs) are a class of short single-stranded non-coding endogenous RNAs and act as negative regulators of gene expression by inhibiting mRNA translation or promoting mRNA degradation.^{15, 16} Although the function of miRNAs has been widely studied in apoptosis, development, differentiation and proliferation, few works have been focused on miRNAs in the mitochondrial network regulation. It has been reported that miR-30b targets to p53 and inhibits mitochondrial fission.¹⁷ In addition, other miRNAs also affect the function of mitochondria by targeting to mitochondrial calcium uniporter.¹⁸ The study of miRNA function in mitochondria may shed new light on the machinery that underlies mitochondrial regulation.This study unveils that PHB1 is involved in the regulation of mitochondrial network in cardiomyocytes. PHB1 inhibits mitochondrial fission and apoptosis in cardiomyocytes. In addition, PHB1 transgenic mice exhibit a reduced myocardial infarction sizes upon myocardial ischemia injury in vivo. In searching for the mechanism by which PHB1 is downregulated under pathologic condition, we identify miR-361 participates in the suppression of PHB1 translation. MiR-361 initiates mitochondrial fission, apoptosis and myocardial infarction through downregulating PHB1. Our results reveal a novel mitochondrial regulating model, which is composed of miR-361 and PHB1. Modulation of their levels may represent a novel approach for interventional treatment of myocardial infarction and heart failure.     

The multifunctional RNA-binding protein hnRNP A1 is required for processing of miR-18a

hnRNP A1 is an RNA-binding protein involved in various aspects of RNA processing. Use of an in vivo cross-linking and immunoprecipitation protocol to find hnRNP A1 RNA targets resulted in the identification of a microRNA (miRNA) precursor, pre-miR-18a. This microRNA is expressed as part of a cluster of intronic RNAs, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92, and potentially acts as an oncogene. Here we show that hnRNP A1 binds specifically to the primary RNA sequence pri-miR-18a before Drosha processing. HeLa cells depleted of hnRNP A1 have reduced in vitro processing activity with pri-miR-18a and also show reduced abundances of endogenous pre-miR-18a. Furthermore, we show that hnRNP A1 is required for miR-18a-mediated repression of a target reporter in vivo. These results underscore a previously uncharacterized role for general RNA-binding proteins as auxiliary factors that facilitate the processing of specific miRNAs.     

LncRNA CACNA1G-AS1 facilitates hepatocellular carcinoma progression through the miR-2392/C1orf61 pathway

Emerging studies have indicated that long noncoding RNAs (lncRNAs) possess various functions in initiating human cancers. However, the role of lncRNAs in hepatocellular carcinoma (HCC) still remains ill understood. In this study, we sought to investigate the role of lncRNA CACNA1G-AS1 in HCC progression. Through bioinformatics analysis, we found that CACNA1G-AS1 expression was significantly upregulated in HCC tissues compared with that in the adjacent normal tissues. Moreover, CACNA1G-AS1 upregulation indicated poor prognosis in HCC patients. Knockdown of CACNA1G-AS1 attenuated the proliferation, migration, and invasion of HCC cells. Additionally, decreased expression of CACNA1G-AS1 prevented epithelial–mesenchymal transition. In vivo assay also showed that CACNA1G-AS1 silencing HCC cells have smaller tumor volumes and weights. Further investigations demonstrated that CACNA1G-AS1 worked as a competing endogenous RNA to bind microRNA-2392 (miR-2392) and thereby alleviate the repression of the downstream target C1orf61. Collectively, CACNA1G-AS1 promotes HCC progression through regulating the miR-2392/C1orf61 pathway.     

LncRNA882 regulates leukemia inhibitory factor (LIF) by sponging miR-15b in the endometrial epithelium cells of dairy goat

Despite the fact that long noncoding RNAs (lncRNAs) play roles in almost all biological processes, little is known about their biological function in the endometrium during the formation of endometrial receptivity. In this study, a comprehensive analysis of lncRNAs in goat endometrial tissues on Day 5 (prereceptive endometrium, PE) and Day 15 (receptive endometrium, RE) of pregnancy was performed by using RNA-Seq. As a result, 668 differentially expressed lncRNAs (DELs) were found between the PE and RE. Further study showed that lncRNA882, regulated by estrogen (E2) and progestin (P4), could act as competing endogenous RNAs (ceRNAs) for miR-15b, which inhibited the expression of transforming growth factor-b-activated kinase 1 binding protein 3 (TAB3) and then indirectly regulated the level of leukemia inhibitory factor (LIF). This was helpful for the formation of endometrial receptivity in dairy goats. In conclusion, we elucidated the endometrium lncRNA profiles of PE and RE in dairy goats; lncRNA882 acted as a ceRNA for miR-15b and then indirectly regulated the level of LIF in goat endometrial epithelium cells. Thus, this study helped us to better understand the molecular regulation of endometrial receptivity in dairy goats.     

LncRNA SNHG20 contributes to cell proliferation and invasion by upregulating ZFX expression sponging miR-495-3p in gastric cancer

Long noncoding RNAs (lncRNAs) exert key regulators in cancer development and progression. The functional significance of lncRNA small nucleolar RNA host gene 20 (SNHG20) was reported in gastric cancer (GC); however, the underlying molecular mechanism in GC development is largely unknown. Here, our results showed that the lncRNA SNHG20 expression was significantly higher in GC tissues compared with adjacent normal tissues by quantitative real-time PCR (qRT-PCR) analysis. Higher lncRNA SNHG20 expression was highly associated with tumor size and lymphatic metastasis of patients. Patients with higher lncRNA SNHG20 expression predicted a short disease-free survival (DFS) and overall survival (OS). Furthermore, lncRNA SNHG20 expression negatively associated with miR-495-3p expression and regulated miR-495-3p expression. Function assays confirmed that lncRNA SNHG20 knockdown using RNA interference suppressed cell proliferation and invasion of GC by negatively regulating miR-495-3p expression. Moreover, we demonstrated that lncRNA SNHG20 inhibited zinc finger protein X-linked (ZFX) expression by negatively miR-495-3p expression in GC cells. In vivo, the current study also indicated that lncRNA SNHG20 knockdown reduced the tumor growth by downregulating ZFX expression. Thus, our results implied that inhibition of SNHG20/miR-495-3p/ZFX axis may provide valuable target for GC treatment.     

利用RIP-Seq技术鉴定HepG2细胞中与HuR蛋白结合的lncRNAs

长链非编码RNAs（long noncoding RNAs, lncRNAs）可在表观遗传水平、转录水平和转录后水平调节基因的表达,对细胞功能起着重要的调节作用。RNA结合蛋白可与很多的RNA结合,并在转录后水平发挥重要的调节作用。然而,RNA结合蛋白是否可以在细胞内广泛结合lncRNAs对其发挥调节作用,仍需进一步证实。本研究通过RNA结合蛋白免疫沉淀技术联合高通量测序（RNA binding protein immunoprecipitation-high throughput sequencing, RIP-Seq）的方法在人肝癌细胞株HepG2中,鉴定与人抗原R（human antigen R, HuR）蛋白相结合的lncRNA分子,并进行了初步的验证。首先,通过HuR-RIP实验分离与HuR蛋白结合的RNA分子,然后高通量测序及生物信息学分析。根据分析结果,鉴定出HepG2细胞中361条与HuR蛋白结合的lncRNAs分子,包括基因间lncRNA（large intergenic noncoding RNA, LincRNA）、内含子lncRNA、与编码基因正义链有重叠的lncRNA和与编码基因反义链有重叠lncRNA（antisense lncRNA）等。并进一步通过RIP-qPCR技术,对其中20条LincRNA分子进行了定量检测,验证测序结果。在HepG2细胞中敲低HuR基因表达,发现这些LincRNA分子中,11条LincRNA分子表达水平显著降低（P＜0.05）,2条LincRNA显著升高（P＜0.05）,剩余7条LincRNA表达量未发生变化（P＞0.05）。本研究结果说明,HuR在细胞内可以广泛结合lncRNA分子,并且可能对结合的lncRNA分子的表达量产生影响,这也为进一步研究这些lncRNA的功能和HuR调控网络的研究提供了基础。     

Adaptor proteins in long noncoding RNA biology

Long noncoding RNAs (lncRNAs) are a heterogeneous class of noncoding RNAs that have gained increasing attention due to their vital roles in the regulation of diverse cellular processes. Because lncRNAs are generally expressed at low levels, are poorly conserved, and can act via diverse mechanisms, investigating the molecular mechanisms by which lncRNAs act is challenging. Similar to mRNAs, lncRNAs bind to RNA-binding proteins (RBPs) and in some cases, have been shown to regulate the activity of the RBP they bind to. Furthermore, recent studies have shown that some lncRNAs directly bind to a specific RBP that, in turn, forms a complex with other proteins that mediate the effects of the lncRNA. We termed such RBPs as adaptor proteins because they function as adaptors to recruit other proteins that indirectly associate with the lncRNA. Here, we discuss the emerging roles of adaptor proteins in lncRNA function and propose mechanistic scenarios and strategies to identify adaptor proteins that could play vital roles in the biology of a lncRNA. This article is part of a Special Issue entitled: ncRNA in control of gene expression edited by Kotb Abdelmohsen.