Identification and detection of mecciRNAs.

Mitochondria participate in series of metabolic processes and cellular events. It is widely known that only 13 proteins are encoded by the mammalian mitochondria genome. However, it is not acknowledged until recently that mitochondrial genomes encode hundreds of circular RNAs, named as mecciRNAs. Some of these mecciRNAs can serve as molecular chaperones to help folding nuclear-encoded proteins and facilitating their mitochondrial entrance. As a novel type of circular RNAs, functions and characteristics of mecciRNAs are waiting for further exploration and methods for mecciRNA studies need to be improved. Here, we describe detailed methods for mitochondrial RNA isolation and mecciRNA detection. In addition, we present effective mecciRNA overexpression and knockdown strategies for future functional studies of mecciRNAs.

Liu X ，Yang Y ，Shan G 《-》

Identification of mecciRNAs and their roles in the mitochondrial entry of proteins.

Liu X ，Wang X ，Li J ，Hu S ，Deng Y ，Yin H ，Bao X ，Zhang QC ，Wang G ，Wang B ，Shi Q ，Shan G ... -  《-》

Emerging functions of mitochondria-encoded noncoding RNAs.

Mitochondria, organelles that harbor their own circular genomes, are critical for energy production and homeostasis maintenance in eukaryotic cells. Recent studies discovered hundreds of mitochondria-encoded noncoding RNAs (mt-ncRNAs), including novel subtypes of mitochondria-encoded circular RNAs (mecciRNAs) and mitochondria-encoded double-stranded RNAs (mt-dsRNAs). Here, we discuss the emerging field of mt-ncRNAs by reviewing their expression patterns, biogenesis, metabolism, regulatory roles, and functional mechanisms. Many mt-ncRNAs have regulatory roles in cellular physiology, and some are associated with, or even act as, causal factors in human diseases. We also highlight developments in technologies and methodologies and further insights into future perspectives and challenges in studying these noncoding RNAs, as well as their potential biomedical applications.

Ren B ，Guan MX ，Zhou T ，Cai X ，Shan G ... -  《TRENDS IN GENETICS》

The potential of mecciRNA in hepatic stellate cell to regulate progression of nonalcoholic hepatitis.

Nonalcoholic steatohepatitis (NASH) occupies a substantial proportion of chronic liver disease worldwide, of which pathogenesis needs further research. Recent studies have demonstrated the significant roles of circular RNAs (circRNAs) in NASH, while the function of a novel type of circRNAs, namely mitochondria-encoded circRNAs (mecciRNAs), remains elusive. Therefore, we aimed to investigate their potential to regulate the progression of NASH in this study. GSE134146 was used to screen for differentially expressed mecciRNAs in NASH, while GSE46300 was used to identify NASH-related genes. To establish the mecciRNA-miRNA-mRNA networks, circMINE and miRNet databases were used for predicting downstream targets. Then, consensus clustering analysis was used to determine immune subtypes of NASH. Finally, we successfully validated our findings in vitro (LPS-treated hepatic stellate cells [HSCs]) and in vivo (MCD-diet mice) NASH models. We confirmed that circRNomics balance is disrupted in HSCs of NASH, while two mecciRNAs (hsa_circ_0089761 and hsa_circ_0089763) could function as competing for endogenous RNAs (ceRNAs) to regulate fibrosis-related signals. Furthermore, we constructed two ceRNA networks based on mecciRNAs for the first time. Cell and animal NASH models validated our findings that c-MYC and SMAD2/3 were upregulated in HSCs, while THBS1 and p-STAT3 were upregulated in hepatocytes. Moreover, we identified 21 core genes by overlapping the differentially expressed genes (NASH vs. Normal) with mecciRNA-targeted genes. According to their expression profiles, NASH patients could be divided in 2 different clusters, in which proinflammatory signals (TNF and IL-17 pathways) are significantly activated in Cluster 1. We successfully established two novel mecciRNA-miRNA-mRNA networks in HSCs and hepatocytes, which were further confirmed by in vitro and in vivo models. Meanwhile, the novel immunotyping model revealed the heterogeneity of NASH, thereby might guiding treatment options. Altogether, our study brought a distinct perspective on the relationship between mecciRNAs and NASH.

Liu B ，Tian Y ，He J ，Gu Q ，Jin B ，Shen H ，Li W ，Shi L ，Yu H ，Shan G ，Cai X ... -  《Journal of Translational Medicine》

p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation.

Metabolic reprogramming sustains tumorigenesis and aggressiveness of neuroblastoma (NB), the most common extracranial malignancy in childhood, while underlying mechanisms and therapeutic approaches still remain elusive. Circular RNAs (circRNAs) were validated by Sanger sequencing. Co-immunoprecipitation, mass spectrometry, chromatin immunoprecipitation (ChIP) sequencing, and RNA sequencing assays were applied to explore protein interaction and target genes. Gene expression regulation was observed by ChIP, dual-luciferase reporter, real-time quantitative RT-PCR, and western blot assays. Gain- and loss-of-function studies were performed to observe the impacts of circRNA-encoded protein and its partners on the lipid metabolism, mitochondrial activity, growth, invasion, and metastasis of NB cells. A novel 113-amino acid protein (p113) of CUT-like homeobox 1 (CUX1) was identified in NB cells treated by serum deprivation. Further validating studies revealed that nuclear p113 was encoded by circRNA of CUX1, and promoted the lipid metabolic reprogramming, mitochondrial activity, proliferation, invasion, and metastasis of NB cells. Mechanistically, p113 interacted with Zuotin-related factor 1 (ZRF1) and bromodomain protein 4 (BRD4) to form a transcriptional regulatory complex, and mediated the transactivation of ZRF1/BRD4 in upregulating ALDH3A1, NDUFA1, and NDUFAF5 essential for conversion of fatty aldehydes into fatty acids, fatty acid β-oxidation, and mitochondrial complex I activity. Administration of an inhibitory peptide blocking p113-ZRF1 interaction suppressed the tumorigenesis and aggressiveness of NB cells. In clinical NB cases, high expression of p113, ZRF1, or BRD4 was associated with poor survival of patients. These results indicate that p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation.

Yang F ，Hu A ，Guo Y ，Wang J ，Li D ，Wang X ，Jin S ，Yuan B ，Cai S ，Zhou Y ，Li Q ，Chen G ，Gao H ，Zheng L ，Tong Q ... -  《Molecular Cancer》