RNA-RNA interactions (RRIs) can dictate RNA molecules to form intricate higher-order structures and bind their RNA substrates in diverse biological processes. To elucidate the function, binding specificity, and regulatory mechanisms of various RNA molecules, especially the vast repertoire of non-coding RNAs, advanced technologies and methods that globally map RRIs are extremely valuable. In the past decades, many state-of-the-art technologies have been developed for this purpose. This review focuses on those high-throughput technologies for the global mapping of RRIs. We summarize the key concepts and the pros and cons of different technologies. In addition, we highlight the novel biological insights uncovered by these RRI mapping methods and discuss the future challenges for appreciating the crucial roles of RRIs in gene regulation across bacteria, viruses, archaea, and mammals.

Granulosa cells (GCs) are essential for follicular development, and long non-coding RNAs (LncRNAs) are known to support the maintenance of this process and hormone synthesis in mammals. Nevertheless, the regulatory roles of these lncRNAs within sheep follicular GCs remain largely unexplored. This study delved into the influence of a Loc105611671, on the proliferation and steroid hormone synthesis of sheep ovarian GCs and the associated target genes in vitro. Cell Counting Kit-8 (CCK-8) gain-of-function experiments indicated that overexpression of Loc105611671 significantly boosted GCs proliferation, along with estrogen (E2) and progesterone (P4) levels. Further mechanistic scrutiny revealed that Loc105611671 is primarily localized within the cytoplasm of ovarian granulosa cells and engages in molecular interplay with CDC42. This interaction results in the upregulation of CDC42 protein expression. Moreover, it was discerned that increased CDC42 levels contribute to augmented proliferation of follicular granulosa cells and the secretion of E2 and P4. Experiments involving co-transfection elucidated that the concurrent overexpression of CDC42 and Loc105611671 acted synergistically to potentiate these effects. These findings provide insights into the molecular underpinnings of fecundity in ovine species and may inform future strategies for enhancing reproductive outcomes.

SARS-CoV-2 RNA interacts with host factors to suppress interferon responses and simultaneously induces cytokine release to drive the development of severe coronavirus disease 2019 (COVID-19). However, how SARS-CoV-2 hijacks host RNAs to elicit such imbalanced immune responses remains elusive. Here, we analyzed SARS-CoV-2 RNA in situ structures and interactions in infected cells and patient lung samples using RIC-seq. We discovered that SARS-CoV-2 RNA forms 2,095 potential duplexes with the 3\' UTRs of 205 host mRNAs to increase their stability by recruiting RNA-binding protein YBX3 in A549 cells. Disrupting the SARS-CoV-2-to-host RNA duplex or knocking down YBX3 decreased host mRNA stability and reduced viral replication. Among SARS-CoV-2-stabilized host targets, NFKBIZ was crucial for promoting cytokine production and reducing interferon responses, probably contributing to cytokine storm induction. Our study uncovers the crucial roles of RNA-RNA interactions in the immunopathogenesis of RNA viruses such as SARS-CoV-2 and provides valuable host targets for drug development.

The elaborate interplay of coding and noncoding factors governs muscle growth and development. Here, we reported a mutual activation between long noncoding RNA (lncRNA) H19 and MyoD (myogenic determination gene number 1) in the muscle process. We successfully cloned the two isoforms of goat H19, which were significantly enriched and positively correlated with MyoD transcripts in skeletal muscles or differentiating muscle satellite cells (MuSCs). To systematically screen genes altered by H19, we performed RNA-seq using cDNA libraries of differentiating H19-deficiency MuSCs and consequently anchored MyoD as the critical genes in mediating H19 function. Intriguingly, some transcripts of MyoD and H19 overlapped in the cytoplasm, which was dramatically damaged when the core complementary nucleotides were mutated. Meanwhile, MyoD RNA successfully pulled down H19 in MS2-RIP experiments. Furthermore, HuR could bind both H19 and MyoD transcripts, while H19 or its truncated mutants successfully stabilized MyoD mRNA, with or without HuR deficiency. In turn, novel functional MyoD protein-binding sites were identified in the promoter and exons of the H19 gene. Our results suggest that MyoD activates H19 transcriptionally, and RNA-RNA hybridization is critical for H19-promoted MyoD expression, which extends our knowledge of the hierarchy of regulatory networks in muscle growth.

Intracerebral hemorrhage (ICH) can usually cause severe neuroinflammation and blood-brain barrier (BBB) damage. Previous studies supported the important role of long non-coding RNAs (lncRNAs) in ICH treatment. This study aimed to explore the effect of lncRNA FGD5 antisense RNA 1 (FGD5-AS1) on ICH and its potential molecular mechanisms. C57BL/6 mice were injected with collagenase VII to establish an ICH mice model. In addition, brain cerebral microvascular endothelial cells (BMVECs) were treated by oxygen-glucose deprivation (OGD)/hemin to simulate ICH. RT-qPCR revealed that FGD5-AS1 was upregulated in serum of ICH patients and mice and in OGD/hemin-treated BMVECs. Luciferase reporter gene and pull-down assays predicted and verified that FGD5-AS1 bound to miR-6838-5p, and VEGFA was a target of miR-6838-5p. FGD5-AS1 knockdown decreased the inflammatory factor contents in brain tissues and BMVECs. FGD5-AS1 overexpression inhibited cell proliferation, invasion and tight junction protein levels, and promoted apoptosis, increased the permeability of BBB and secretion of pro-inflammatory factors. In addition, miR-6838-5p knockdown reversed the inhibitory effect of FGD5-AS1 knockdown on the PI3K/Akt signaling pathway. In conclusion, FGD5-AS1 may act as an important regulator to promote apoptosis, cell permeability and inflammatory response of BMVECs via the miR-6838-5p/VEGFA axis in ICH mice.

Renal cell carcinoma (RCC) is one of the most common renal malignancies in the urinary system. Numerous studies have demonstrated that miRNAs can regulate tumorigenesis and progression. This study aims to investigate the role and regulatory mechanism of miR-6838-5p in RCC. Our study confirmed that miR-6838-5p was upregulated in human RCC tissues (30/42, 77.43%, P < 0.01) and RCC cell lines (P < 0.05) compared to adjacent non-neoplastic tissues and normal renal epithelial cells. In vitro, overexpression of miR-6838-5p enhanced cell proliferation and invasion in human RCC cell lines (ACHN and 786-O), which were detected by CCK-8, Transwell and Colony formation assays (P < 0.05), and knockdown of miR-6838-5p suppressed cell proliferation and invasion (P < 0.05). Results of Bioinformatics analysis combined with Dual-luciferase reporter gene assay demonstrated that miR-6838-5p could bind to Cyclin D binding myb-like transcription factor 1 (DMTF1). In addition, RT-qPCR and Western blotting confirmed that DMTF1 was downregulated in RCC tissues and cell lines. Meanwhile, it was demonstrated that overexpression of miR-6838-5p inhibited DMTF1 level in ACHN cells. Next, we confirmed that DMTF1 overexpression reversed the inhibitory effects of overexpression of miR-6838-5p on phosphatase and tensin homolog (PTEN), tumor protein 53(p53), murine double minute 2 (MDM2) and alternative reading frame (ARF) protein levels in the ARF-p53 signaling pathway. In conclusion, our research showed that miR-6838-5p enhanced the proliferation and invasion of RCC cells by inhibiting the DMTF1/ARF-p53 axis.

Influenza A virus (IAV) contains a genome with eight single-stranded, negative-sense RNA segments that encode 17 proteins. During its assembly, all eight separate viral RNA (vRNA) segments are incorporated into virions in a selective manner. Evidence suggested that the highly selective genome packaging mechanism relies on RNA-RNA or protein-RNA interactions. The specific structures of each vRNA that contribute to mediating the packaging of the vRNA into virions have been described and identified as packaging signals. Abundant research indicated that sequences required for genome incorporation are not series and are varied among virus genotypes. The packaging signals play important roles in determining the virus replication, genome incorporation and genetic reassortment of influenza A virus. In this review, we discuss recent studies on influenza A virus packaging signals to provide an overview of their characteristics and functions.

Tetralogy of Fallot (TOF) is the most common complex congenital heart disease (CHD) with uncertain cause. Although long non-coding RNAs (lncRNAs) have been implicated in heart development and several CHDs, their role in TOF is not well understood. This study aimed to investigate how dysregulated lncRNAs contribute to TOF. Using Gene Expression Omnibus data mining, bioinformatics analysis and clinical heart tissue sample detecting, we identified a novel antisense lncRNA TBX5-AS1:2 with unknown function that was significantly down-regulated in injured cardiac tissues from TOF patients. LncRNA TBX5-AS1:2 was mainly located in the nucleus of the human embryonic kidney 293 (HEK293T) cells and formed an RNA-RNA double-stranded structure in the overlapping region with its sense mRNA T-box transcription factor 5 (TBX5), which is an important regulator in heart development. Knock-down of lncRNA TBX5-AS1:2 via promoter hypermethylation reduced TBX5 expression at both the mRNA and protein levels by affecting its mRNA stability through RNA-RNA interaction. Moreover, lncRNA TBX5-AS1:2 knock-down inhibited the proliferation of HEK293T cells. In conclusion, these results indicated that lncRNA TBX5-AS1:2 may be involved in TOF by affecting cell proliferation by targeting TBX5.

Accumulating evidences highlight the critical roles of long noncoding RNAs (lncRNAs) in a variety of cancers. LncRNA PXN-AS1-L was previously shown to exert oncogenic roles in hepatocellular carcinoma. However, the expression, role, and molecular mechanism of PXN-AS1-L in nasopharyngeal carcinoma (NPC) malignancy remain unknown. Here, we determined that PXN-AS1-L is upregulated in NPC tissues and cell lines. Increased expression of PXN-AS1-L predicts worse prognosis of NPC patients. PXN-AS1-L overexpression promotes NPC cell proliferation, migration, and invasion in vitro, and NPC tumor growth in vivo. PXN-AS1-L silencing suppresses NPC cell proliferation, migration, and invasion in vitro. Mechanistically, PXN-AS1-L directly interacts with SAPCD2 mRNA 3\'-untranslated region, prevents the binding of microRNAs-AGO silencing complex to SAPCD2 mRNA, and upregulates the mRNA and protein level of SAPCD2. SAPCD2 is also increased in NPC tissues. The expression of SAPCD2 is significantly positively associated with that of PXN-AS1-L in NPC tissues. Gain-of-function and loss-of-function experiments demonstrated that SAPCD2 also promotes NPC cell proliferation, migration, and invasion. Furthermore, depletion of SAPCD2 significantly reverses the roles of PXN-AS1-L in promoting NPC cell proliferation, migration, and invasion in vitro, and NPC tumor growth in vivo. In conclusion, lncRNA PXN-AS1-L is upregulated in NPC and promoted NPC malignancy by upregulating SAPCD2 via direct RNA-RNA interaction.

Zika virus (ZIKV) is an Aedes mosquitoes-transmitted flavivirus, and its infection may cause severe neurological diseases. A genetically stable infectious clone is essential for ZIKV research, however the toxicity and instability of the viral cDNA in bacteria potentially due to its bacterial promoter activity are major challenges. Here, we constructed a full-length cDNA clone for isolate ZG01 by introducing non-coding changes T1865C/A1868G to reduce the bacterial promoter activity. Wild-type and recombinant ZG01 were highly attenuated in Vero cells, thus we serially passaged wild-type ZG01 through neonatal mice and Vero cells to generate high-titer virus, from which four mutations (4m, C2178T/G2913A/T4991C/T10561C) were identified. Addition of 4m greatly enhanced the infectivity, as ZG01_4m released ZIKV of 107.0-107.5 plaque-forming unit (PFU)/ml in infected Vero and A549 cells. ZG01_4m resembled the infectivity of high-titer ZG01 in vitro and in vivo. Notably, ZG01_4m plasmid was genetically stable after multiple rounds of transformation-purification in bacteria. Using ZG01_4m, we identified a potential RNA-RNA interaction between 5\'UTR and 3\'UTR and demonstrated that the nucleotides involved were essential for ZIKV production. The genetically stable ZG01 cDNA clone provides a reliable tool for the study of this important virus, and the strategy used here is feasible for the development of reverse genetics systems for other ZIKV isolates and related flaviviruses.