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Liver development is a highly complex process that is regulated by the orchestrated interplay of epigenetic regulators, transcription factors, and microRNAs (miRNAs). Owing to the lack of global in vivo targets of all miRNAs during liver development, the mechanisms underlying the dynamic control of hepatocyte differentiation by miRNAs remain elusive. Here, using Argonaute (Ago) high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) in the mouse liver at different developmental stages, we characterized massive Ago-binding RNAs and obtained a genome-wide map of liver miRNA-mRNA interactions. The dynamic changes of five clusters of miRNAs and their potential targets were identified to be differentially involved at specific stages, a dozen of high abundant miRNAs and their epigenetic regulation by super-enhancer were found during liver development. Remarkably, miR-122, a liver-specific and most abundant miRNA in newborn and adult livers, was found by its targetome and pathway reporter analyses to regulate the Hippo pathway, which is crucial for liver size control and homeostasis. Mechanistically, we further demonstrated that miR-122 negatively regulates the outcomes of the Hippo pathway transcription factor TEAD by directly targeting a number of hippo pathway regulators, including the coactivator TAZ and a key factor of the phosphatase complex PPP1CC, which contributes to the dephosphorylation of YAP, another coactivator downstream of the Hippo pathway. This study identifies for the first time the genome-wide miRNA targetomes during mouse liver development and demonstrates a novel mechanism of terminal differentiation of hepatocytes regulated by the miR-122/Hippo pathway in a coordinated manner. As the Hippo pathway plays important roles in cell proliferation and liver pathological processes like inflammation, fibrosis, and hepatocellular carcinoma (HCC), our study could also provide a new insight into the function of miR-122 in liver pathology.

Copy number variations (CNVs), which can affect the role of long non-coding RNAs (lncRNAs), are important genetic changes seen in some malignant tumors. We analyzed lncRNAs with CNV to explore the relationship between lncRNAs and prognosis in bladder cancer (BLCA). Messenger RNA (mRNA) expression levels, DNA methylation, and DNA copy number data of 408 BLCA patients were subjected to integrative bioinformatics analysis. Cluster analysis was performed to obtain different subtypes and differently expressed lncRNAs and coding genes. Weighted gene co-expression network analysis (WGCNA) was performed to identify the co-expression gene and lncRNA modules. CNV-associated lncRNA data and their influence on cancer prognosis were assessed with Kaplan-Meier survival curve. Multi-omics integration analysis revealed five prognostic lncRNAs with CNV, namely NR2F1-AS1, LINC01138, THUMPD3-AS1, LOC101928489,and TMEM147-AS1,and a risk-score signature related to overall survival in BLCA was identified. Moreover, validated results in another independent Gene Expression Omnibus (GEO) dataset, GSE31684, were consistent with these results. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the mitogen-activated protein kinase (MAPK) signaling pathway, focal adhesion pathway, and Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling pathway were enriched in a high-risk score pattern, suggesting that imbalance in these pathways is closely related to tumor development. We revealed the prognosis-related lncRNAs by analyzing the expression profiles of lncRNAs and CNVs, which can be used as prognostic biomarkers for BLCA.

Skeletal muscle differentiation is a highly coordinated process that involves many cellular signaling pathways and microRNAs (miRNAs). A group of muscle-specific miRNAs has been reported to promote myogenesis by suppressing key signaling pathways for cell growth. However, the functional role and regulatory mechanism of most non-muscle-specific miRNAs with stage-specific changes during differentiation are largely unclear. Here, we describe the functional characterization of miR-101a/b, a pair of non-muscle-specific miRNAs that show the largest change among a group of transiently upregulated miRNAs during myogenesis in C2C12 cells. The overexpression of miR-101a/b inhibits myoblast differentiation by suppressing the p38/MAPK, Interferon Gamma, and Wnt pathways and enhancing the C/EBP pathway. Mef2a, a key protein in the p38/MAPK pathway, was identified as a direct target of miR-101a/b. Interestingly, we found that the long non-coding RNA (lncRNA) Malat1, which promotes muscle differentiation, interacts with miR-101a/b, and this interaction competes with Mef2a mRNA to relieve the inhibition of the p38/MAPK pathway during myogenesis. These results uncovered a “braking” role in differentiation of transiently upregulated miRNAs and provided new insights into the competing endogenous RNA (ceRNA) regulatory mechanism in myoblast differentiation and myogenesis.

Eukaryotic genomes encode thousands of small and large non-coding RNAs (ncRNAs). However, the expression, functions and evolution of these ncRNAs are still largely unknown. In this study, we have updated deepBase to version 3.0 (deepBase v3.0, http://rna.sysu.edu.cn/deepbase3/index.html), an increasingly popular and openly licensed resource that facilitates integrative and interactive display and analysis of the expression, evolution, and functions of various ncRNAs by deeply mining thousands of high-throughput sequencing data from tissue, tumor and exosome samples. We updated deepBase v3.0 to provide the most comprehensive expression atlas of small RNAs and lncRNAs by integrating ∼67 620 data from 80 normal tissues and ∼50 cancer tissues. The extracellular patterns of various ncRNAs were profiled to explore their applications for discovery of noninvasive biomarkers. Moreover, we constructed survival maps of tRNA-derived RNA Fragments (tRFs), miRNAs, snoRNAs and lncRNAs by analyzing >45 000 cancer sample data and corresponding clinical information. We also developed interactive webs to analyze the differential expression and biological functions of various ncRNAs in ∼50 types of cancers. This update is expected to provide a variety of new modules and graphic visualizations to facilitate analyses and explorations of the functions and mechanisms of various types of ncRNAs.

Kinetoplastid flagellates are known for several unusual features, one of which is their complex mitochondrial genome, known as kinetoplast (k) DNA, composed of mutually catenated maxi- and minicircles. Trypanosoma lewisi is a member of the Stercorarian group of trypanosomes which is, based on human infections and experimental data, now considered a zoonotic pathogen. By assembling a total of 58 minicircle classes, which fall into two distinct categories, we describe a novel type of kDNA organization in T. lewisi. RNA-seq approaches allowed us to map the details of uridine insertion and deletion editing events upon the kDNA transcriptome. Moreover, sequencing of small RNA molecules enabled the identification of 169 unique guide (g) RNA genes, with two differently organized minicircle categories both encoding essential gRNAs. The unprecedented organization of minicircles and gRNAs in T. lewisi broadens our knowledge of the structure and expression of the mitochondrial genomes of these human and animal pathogens. Finally, a scenario describing the evolution of minicircles is presented.

Charcoal-stripped fetal bovine serum (CS-FBS) is frequently used in studies on hormone-responsive cancers to provide hormone-free cell culture conditions. CS-FBS may influence the growth of cancer cells; however, the underlying mechanisms remain unclear. In this study, we aimed to clarify the effects of CS-FBS on distinct subtypes of breast cancer cells. We found that the crucial oncoprotein c-Myc was significantly inhibited in estrogen receptor alpha (ER-α)-positive breast cancer cells when cultured in CS-FBS-supplemented medium, but it was not suppressed in ER-α-negative cells. The addition of 17β-estradiol (E2) to CS-FBS-supplemented medium rescued the CS-FBS-induced inhibition of c-Myc, while treatment with 5α-dihydrotestosterone (DHT) suppressed c-Myc expression. Our data demonstrated that CS-FBS may impede the growth of ER-α-positive breast cancer cells via c-Myc inhibition, and this was possibly due to the removal of estrogen. These results highlighted that the core drivers of c-Myc expression were subtype-specific depending on the distinct cell context and special caution should be exercised when using CS-FBS in studies of hormone-responsive cancer cells.

tRNA-derived small RNA (tsRNA), a novel type of regulatory small noncoding RNA, plays an important role in physiological and pathological processes. However, the understanding of the functional mechanism of tsRNAs in cells and their role in the occurrence and development of diseases is limited. Here, we integrated multiomics data such as transcriptome, epitranscriptome, and targetome data, and developed novel computer tools to establish tsRFun, a comprehensive platform to facilitate tsRNA research (http://rna.sysu.edu.cn/tsRFun/ or http://biomed.nscc-gz.cn/DB/tsRFun/). tsRFun evaluated tsRNA expression profiles and the prognostic value of tsRNAs across 32 types of cancers, identified tsRNA target molecules utilizing high-throughput CLASH/CLEAR or CLIP sequencing data, and constructed the interaction networks among tsRNAs, microRNAs, and mRNAs. In addition to its data presentation capabilities, tsRFun offers multiple real-time online tools for tsRNA identification, target prediction, and functional enrichment analysis. In summary, tsRFun provides a valuable data resource and multiple analysis tools for tsRNA investigation.

The low incidence rates of prostatic extra-gastrointestinal stromal tumors (EGIST), combined with the lack of published guidelines on its treatment, often results in its misdiagnosis and challenges in the treatment of patients, even in cases with high-risk factors. The present case study reported a 65-years-old Chinese male patient, who presented with intermittent hematuria and lower urinary tract symptoms for three months. The colonoscopy results revealed no gastrointestinal lesions; however, a core biopsy diagnosed an EGIST, which subsequently underwent radical prostatocystotomy, standard pelvic lymph node resection, and bricker ileal conduit diversion. The postoperative pathological results suggested a high-risk primary prostatic EGIST, according to the aggressive behavior of the GIST. The immunohistochemistry results revealed the positive expression of CD117, DOG1, CD34, androgen receptor AR, prostate-specific antigen (PSA), a 2% Ki-67 index and a positive surgical margin. The whole exome sequencing (WES) results revealed that the patient harbored a single nucleotide mutation in 121 genes and copy number variations in 601 genes, including a defect in c-Kit (in-frame deletion in p.Q556-V560; fold, 17.5%). By compiling the data obtained from the ConsensusPathDB and the drug-gene interaction databases and expert opinions, the patient was prescribed with the personalized drugs (400 mg per day imatinib mesylate and 50 mg per day bicalutamide, which were stopped when the PSA levels remained stable below 0.01 ng/ml) for 18 months follow-up and there were no signs of recurrence. In conclusion, WES identified multiple genomic alterations and the underlying genetic defect in the rare case enabled the evaluation of the prognosis and the decision of potential drug candidates. The underlying mechanism of the substantial genetic variations in the primary prostatic EGIST, as well as the malignant behaviors of the tumor, remain to be investigated.

Rice (Oryza sativa) is a facultative short-day (SD) plant, flowering early under SD and late under long-day (LD) conditions. Ghd7 is a major regulator of flowering time in rice, which strongly delays flowering under LD. Induction of Ghd7 expression by phytochromes has been shown to contribute to photoperiodic regulation of flowering in rice. Here, we show that Ghd7 also is regulated by phytochromes at a post-transcriptional level. We found that constitutive expression of Ghd7 delays flowering in the wild-type (WT) background, but not in the se5 mutant background (deficient in functional phytochromes) under LD and that Ghd7 protein fails to accumulate in the se5 mutant. We also found that co-expressing OsGIGANTEA (OsGI) with Ghd7 causes reduced accumulation of Ghd7 protein and partially suppresses the delayed flowering phenotype in the WT background, suggesting that phytochromes and OsGI play antagonist roles in regulating Ghd7 protein stability and flowering time. We show that OsPHYA, OsPHYB and OsGI could directly interact with Ghd7. Interestingly, OsPHYA and OsPHYB could inhibit the interaction between OsGI and Ghd7, thus helping to stabilize Ghd7 protein. Our results revealed a new level of Ghd7 regulation by phytochromes and OsGI in photoperiodic control of flowering in rice.

The roles of concealed microproteins encoded by long noncoding RNAs (lncRNAs) are gradually being exposed, but their functions in tumorigenesis are still largely unclear. Here, we identify and characterize a conserved 99-amino acid microprotein named KRASIM that is encoded by the putative lncRNA NCBP2-AS2. KRASIM is differentially expressed in normal hepatocytes and hepatocellular carcinoma (HCC) cells and can suppress HCC cell growth and proliferation. Mechanistically, KRASIM interacts and colocalizes with the KRAS protein in the cytoplasm of human HuH-7 hepatoma cells. More importantly, the overexpression of KRASIM decreases the KRAS protein level, leading to the inhibition of ERK signaling activity in HCC cells. These results demonstrate a novel microprotein repressor of the KRAS pathway for the first time and provide new insights into the regulatory mechanisms of oncogenic signaling and HCC therapy.

DNA and histone modifications have notable effects on gene expression1. Being the most prevalent internal modification in mRNA, the N6-methyladenosine (m6A) mRNA modification is as an important post-transcriptional mechanism of gene regulation2–4 and has crucial roles in various normal and pathological processes5–12. However, it is unclear how m6A is specifically and dynamically deposited in the transcriptome. Here we report that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m6A deposition globally. We show that m6A modifications are enriched in the vicinity of H3K36me3 peaks, and are reduced globally when cellular H3K36me3 is depleted. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a crucial component of the m6A methyltransferase complex (MTC), which in turn facilitates the binding of the m6A MTC to adjacent RNA polymerase II, thereby delivering the m6A MTC to actively transcribed nascent RNAs to deposit m6A co-transcriptionally. In mouse embryonic stem cells, phenocopying METTL14 knockdown, H3K36me3 depletion also markedly reduces m6A abundance transcriptome-wide and in pluripotency transcripts, resulting in increased cell stemness. Collectively, our studies reveal the important roles of H3K36me3 and METTL14 in determining specific and dynamic deposition of m6A in mRNA, and uncover another layer of gene expression regulation that involves crosstalk between histone modification and RNA methylation. METTL14 recognizes the trimethyl mark on lysine 36 of histone H3 that directs m6A modifications co-transcriptionally.

The unfolded protein response (UPR) plays important roles in various cells that have a high demand for protein folding, which are involved in the process of cell differentiation and development. Here, we separately knocked down the three sensors of the UPR in myoblasts and found that PERK knockdown led to a marked transformation in myoblasts from a fusiform to a rounded morphology, which suggests that PERK is required for early myoblast differentiation. Interestingly, knocking down PERK induced reprogramming of C2C12 myoblasts into stem-like cells by altering the miRNA networks associated with differentiation and stemness maintenance, and the PERK-ATF4 signaling pathway transactivated muscle differentiation-associated miRNAs in the early stage of myoblast differentiation. Furthermore, we identified Ppp1cc as a direct target gene of miR-128 regulated by the PERK signaling pathway and showed that its repression is critical for a feedback loop that regulates the activity of UPR-associated signaling pathways, leading to cell migration, cell fusion, endoplasmic reticulum expansion, and myotube formation during myoblast differentiation. Subsequently, we found that the RNA-binding protein ARPP21, encoded by the host gene of miR-128-2, antagonized miR-128 activity by competing with it to bind to the 3′ untranslated region (UTR) of Ppp1cc to maintain the balance of the differentiation state. Together, these results reveal the crucial role of PERK signaling in myoblast maintenance and differentiation and identify the mechanism underlying the role of UPR signaling as a major regulator of miRNA networks during early differentiation of myoblasts.

Background Immune checkpoint inhibitors blocking programmed cell death 1 (PD-1) or programmed cell death ligand 1 (PD-L1) have emerged as effective treatment options for cancer. However, immunotherapy is only effective in a subset of patients. Identifying effective biomarkers to predict the treatment response to PD-1/PD-L1 inhibitors remains an unmet clinical need. Methods This study retrospectively analyzed clinical information and genetic profiling results of 16,013 samples from Chinese patients with various cancer types in order to investigate the prevalence of CD274 (also known as PD-L1) amplification in various cancer types and its association with existing PD-1/PD-L1 biomarkers, including tumor mutational burden (TMB), microsatellite instability (MSI), and PD-L1 expression. Results Amplification of CD274 was identified in 174 samples with an overall prevalence of 1.09% among all cancer types in the cohort. The prevalence of CD274 amplification in different cancer types and histological subtypes of lung cancer was varied, with cervical cancer having the highest prevalence. Distinct distributions of TMB, MSI, and PD-L1 expression between CD274-amplified and wild-type samples were observed in several cancer types as well as among different histological subtypes of lung cancer. Conclusions Although CD274 amplification was only observed in a small proportion of patients, it demonstrated an association with TMB, MSI, and PD-L1 expression in several common cancer types. The molecular features of CD274 in different cancer types are heterogeneous. The role of CD274 amplification as a novel biomarker of PD-1/PD-L1 inhibitors remains to be characterized in future prospective clinical studies.

Although long noncoding RNAs (lncRNAs) have significant tissue specificity, their expression and variability in single cells remain unclear. Here, we developed ColorCells (http://rna.sysu.edu.cn/colorcells/), a resource for comparative analysis of lncRNAs expression, classification and functions in single-cell RNA-Seq data. ColorCells was applied to 167 913 publicly available scRNA-Seq datasets from six species, and identified a batch of cell-specific lncRNAs. These lncRNAs show surprising levels of expression variability between different cell clusters, and has the comparable cell classification ability as known marker genes. Cell-specific lncRNAs have been identified and further validated by in vitro experiments. We found that lncRNAs are typically co-expressed with the mRNAs in the same cell cluster, which can be used to uncover lncRNAs’ functions. Our study emphasizes the need to uncover lncRNAs in all cell types and shows the power of lncRNAs as novel marker genes at single cell resolution.

Hypertrophic growth of cardiomyocytes is one of the major compensatory responses in the heart after physiological or pathological stimulation. Protein synthesis enhancement, which is mediated by the translation of messenger RNAs, is one of the main features of cardiomyocyte hypertrophy. Although the transcriptome shift caused by cardiac hypertrophy induced by different stimuli has been extensively investigated, translatome dynamics in this cellular process has been less studied. Here, we generated a nucleotide-resolution translatome as well as transcriptome data from isolated primary cardiomyocytes undergoing hypertrophy. More than 10,000 open reading frames (ORFs) were detected from the deep sequencing of ribosome-protected fragments (Ribo-seq), which orchestrated the shift of the translatome in hypertrophied cardiomyocytes. Our data suggest that rather than increase the translational rate of ribosomes, the increased efficiency of protein synthesis in cardiomyocyte hypertrophy was attributable to an increased quantity of ribosomes. In addition, more than 100 uncharacterized short ORFs (sORFs) were detected in long noncoding RNA genes from Ribo-seq with potential of micropeptide coding. In a random test of 15 candidates, the coding potential of 11 sORFs was experimentally supported. Three micropeptides were identified to regulate cardiomyocyte hypertrophy by modulating the activities of oxidative phosphorylation, the calcium signaling pathway, and the mitogen-activated protein kinase (MAPK) pathway. Our study provides a genome-wide overview of the translational controls behind cardiomyocyte hypertrophy and demonstrates an unrecognized role of micropeptides in cardiomyocyte biology.

The CREB1 gene encodes a pleiotropic transcription factor that frequently dysregulated in cancers. CREB1 can regulates tumour cell status of proliferation or migration, however, the molecular basis for this switch involvement in cell plasticity has not been fully understood. Here, we show that knocking out CREB1 triggered a remarkable effect of epithelial-mesenchymal transition (EMT) and led to the occurrence of inhibited proliferation and enhanced motility in cancer cells. Mechanistically, CREB1-knockout cells showed arrest in the G0/G1 phase as a result of impaired CREB1-dependent transcription of CCAT1 and E2F1. Interestingly, the competition between the coactivator CBP/p300 for CREB1 and p65 leads to the activation of the NF-κB pathway in cells with CREB1 disrupted, which induces an EMT phenotype and enhances motility. These studies identified previously unknown mechanisms of CREB1 in cell plasticity via its lncRNA and protein effector pathways, revealing an important feature that should be considered in CREB1-targeted tumour therapies.

miR-372/373, a cluster of stem cell-specific microRNAs transactivated by the Wnt pathway, has been reported to be dysregulated in various cancers, particularly colorectal cancer (CRC); however, the unique role of these microRNAs in cancer remains to be discovered. In the present study, we characterized the upregulation in expression of miR-372/373 in CRC tissues from The Cancer Genome Atlas data, and then showed that overexpression of miR-372/373 enhanced the stemness of CRC cells by enriching the CD26/CD24-positive cell population and promoting self-renewal, chemotherapy resistance and the invasive potential of CRC cells. To clarify the mechanism underlying microRNA-induced stemness, we profiled 45 cell signaling pathways in CRC cells overexpressing miR-372/373 and found that stemness-related pathways, such as Nanog and Hedgehog, were upregulated. Instead, differentiation-related pathways, such as NFκB, MAPK/Erk and VDR, were markedly repressed by miR-372/373. Numerous new targets of miR-372/373 were identified, including SPOP, VDR and SETD7, all of which are factors important for cell differentiation. Furthermore, in contrast to the increase in miR-372/373 expression in CRC tissues, the expression levels of SPOP and VDR mRNA were significantly downregulated in these tissues, indicative of the poor differentiation status of CRC. Taken together, our findings suggest that miR-372/373 enhance CRC cell stemness by repressing the expression of differentiation genes. These results provide new insights for understanding the function and mechanisms of stem cell-specific microRNAs in the development of metastasis and drug resistance in CRC.

C-Myc and signal transducer and activator of transcription (STAT) family proteins have been proposed to be important downstream genes of BCR-ABL, which characterizes most cases of chronic myeloid leukemia (CML). Here, we report a c-Myc pathway-targeted screening of seven natural anticancer compounds, in which we identified cryptotanshinone as a highly promising agent for CML therapy. Cryptotanshinone depletes c-Myc in CML by repressing the phosphorylation of STAT5. Decreased viability of K562 cells correlated with p-STAT5 suppression. Unexpectedly, imatinib activates rather than inhibits the phosphorylation of STAT3 in K562 cells. We demonstrated that cryptotanshinone, as a dual inhibitor of p-STAT5 and p-STAT3, can effectively block IL-6-mediated STAT3 activation and reverse BCR-ABL kinase-independent drug resistance. Moreover, we showed that the epigenetic rebalance between decreased BCR-ABL/STAT5/c-Myc and enhanced STAT3/multi-drug resistance (MDR) pathways is characteristic of the cancer stem cell-like property of K562/ADR. Simultaneously suppressing these two pathways using cryptotanshinone proves to be critical for the malignant network redress and MDR reversal of K562/ADR. These studies reveal the dual functions of cryptotanshinone that suppress key oncogenic proliferation and drug-resistant pathways in CML cells by targeting p-STAT5 and p-STAT3, providing a new strategy for CML therapy that takes advantage of natural products.

Cut-like homeobox 1 (CUX1) is a highly conserved homeoprotein that functions as a transcriptional repressor of genes specifying terminal differentiation. We previously showed that liver-specific microRNA-122 (miR-122) regulates the timing of liver development by silencing CUX1 post-transcriptionally. Since the CUX1 protein is expressed in a subset of embryonic tissues, we hypothesized that it is regulated by specific microRNAs (miRNAs) in each cell type during development. Using a large-scale screening method, we identified ten tissue-specific miRNAs from different cell lineages that directly targeted CUX1. An analysis of the interaction between heart-specific microRNA-208a (miR-208a) and CUX1 in the hearts of developing mouse embryos and in P19CL6 cells undergoing cardiac differentiation indicated that CUX1 is regulated by miR-208a during heart development and cardiomyocyte differentiation. Functional analysis of miR-208a in P19CL6 cells using lentiviral-mediated over-expression showed that it regulates the transition between cellular proliferation and differentiation. These results suggest that these tissue-specific miRNAs might play a common role in timing the progression of terminal differentiation of different cell lineages, possibly by silencing the differentiation repressor CUX1.

Precision oncology aims to offer the most appropriate treatments to cancer patients mainly based on their individual genetic information. Genomics has provided numerous valuable data on driver mutations and risk loci; however, it remains a formidable challenge to transform these data into therapeutic agents. Transcriptomics describes the multifarious expression patterns of both mRNAs and non-coding RNAs (ncRNAs), which facilitates the deciphering of genomic codes. In this review, we take breast cancer as an example to demonstrate the applications of these rich RNA resources in precision medicine exploration. These include the use of mRNA profiles in triple-negative breast cancer (TNBC) subtyping to inform corresponding candidate targeted therapies; current advancements and achievements of high-throughput RNA interference (RNAi) screening technologies in breast cancer; and microRNAs as functional signatures for defining cell identities and regulating the biological activities of breast cancer cells. We summarize the benefits of transcriptomic analyses in breast cancer management and propose that unscrambling the core signaling networks of cancer may be an important task of multiple-omic data integration for precision oncology. Keywords: Precision oncology, Transcriptomics, RNA interference, microRNA, Breast cancer

N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic messenger RNAs (mRNAs) and is interpreted by its readers, such as YTH domain-containing proteins, to regulate mRNA fate. Here, we report the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3) as a distinct family of m6A readers that target thousands of mRNA transcripts through recognizing the consensus GG(m6A)C sequence. In contrast to the mRNA-decay-promoting function of YTH domain-containing family protein 2, IGF2BPs promote the stability and storage of their target mRNAs (for example, MYC) in an m6A-dependent manner under normal and stress conditions and therefore affect gene expression output. Moreover, the K homology domains of IGF2BPs are required for their recognition of m6A and are critical for their oncogenic functions. Thus, our work reveals a different facet of the m6A-reading process that promotes mRNA stability and translation, and highlights the functional importance of IGF2BPs as m6A readers in post-transcriptional gene regulation and cancer biology.

Summary: RNA-binding proteins (RBPs) regulate the expression of thousands of transcripts, and some are reported to be involved in human tumorigenesis. However, little is known about the dysregulation of RBPs at the genomic level in human cancers. Here, we conducted comprehensive analyses for expression, somatic copy number alteration (SCNA), and mutation profiles of 1,542 RBPs in ∼7,000 clinical specimens across 15 cancer types. We identified markedly dysregulated RBPs and found that downregulation was a predominant pattern in cancer. Combined with recurrent SCNA data, we identified 76 RBPs as potential drivers. We also discovered a set of 139 RBPs that were significantly mutated in cancers. We confirmed the oncogenic property of six RBPs in colorectal and liver cancer cell lines by using in vitro functional experiments. Our study highlights the potential roles of RBPs in carcinogenesis and lays the groundwork to better understand the functions and mechanisms of RBPs in cancer. : Wang et al. characterize transcriptional and genomic alterations in the landscape of RNA-binding proteins (RBPs) in ∼7,000 clinical samples across 15 human cancer types and experimentally validate the effects of several cancer-related RBPs on cell viability. Keywords: RNA-binding proteins, dysregulation, somatic copy number alterations, mutation profile, cancer driver

Deoxycytidine monophosphate deaminase (dCMP deaminase, DCD) is crucial to the production of dTTP needed for DNA replication and damage repair. However, the effect of DCD deficiency and its molecular mechanism are poorly understood in plants. Here, we isolated and characterized a rice albinic leaf and growth retardation (alr) mutant that is manifested by albinic leaves, dwarf stature and necrotic lesions. Map-based cloning and complementation revealed that ALR encodes a DCD protein. OsDCD was expressed ubiquitously in all tissues. Enzyme activity assays showed that OsDCD catalyses conversion of dCMP to dUMP, and the ΔDCD protein in the alr mutant is a loss-of-function protein that lacks binding ability. We report that alr plants have typical DCD-mediated imbalanced dNTP pools with decreased dTTP; exogenous dTTP recovers the wild-type phenotype. A comet assay and Trypan Blue staining showed that OsDCD deficiency causes accumulation of DNA damage in the alr mutant, sometimes leading to cell apoptosis. Moreover, OsDCD deficiency triggered cell cycle checkpoints and arrested cell progression at the G1/S-phase. The expression of nuclear and plastid genome replication genes was down-regulated under decreased dTTP, and together with decreased cell proliferation and defective chloroplast development in the alr mutant this demonstrated the molecular and physiological roles of DCD-mediated dNTP pool balance in plant development.

More than 100 distinct chemical modifications to RNA have been characterized so far. However, the prevalence, mechanisms and functions of various RNA modifications remain largely unknown. To provide transcriptome-wide landscapes of RNA modifications, we developed the RMBase v2.0 (http://rna.sysu.edu.cn/rmbase/), which is a comprehensive database that integrates epitranscriptome sequencing data for the exploration of post-transcriptional modifications of RNAs and their relationships with miRNA binding events, disease-related single-nucleotide polymorphisms (SNPs) and RNA-binding proteins (RBPs). RMBase v2.0 was expanded with ∼600 datasets and ∼1 397 000 modification sites from 47 studies among 13 species, which represents an approximately 10-fold expansion when compared with the previous release. It contains ∼1 373 000 N6-methyladenosines (m6A), ∼5400 N1-methyladenosines (m1A), ∼9600 pseudouridine (Ψ) modifications, ∼1000 5-methylcytosine (m5C) modifications, ∼5100 2′-O-methylations (2′-O-Me), and ∼2800 modifications of other modification types. Moreover, we built a new module called ‘Motif’ that provides the visualized logos and position weight matrices (PWMs) of the modification motifs. We also constructed a novel module termed ‘modRBP’ to study the relationships between RNA modifications and RBPs. Additionally, we developed a novel web-based tool named ‘modMetagene’ to plot the metagenes of RNA modification along a transcript model. This database will help researchers investigate the potential functions and mechanisms of RNA modifications.

Although thousands of pseudogenes have been annotated in the human genome, their transcriptional regulation, expression profiles and functional mechanisms are largely unknown. In this study, we developed dreamBase (http://rna.sysu.edu.cn/dreamBase) to facilitate the investigation of DNA modification, RNA regulation and protein binding of potential expressed pseudogenes from multidimensional high-throughput sequencing data. Based on ∼5500 ChIP-seq and DNase-seq datasets, we identified genome-wide binding profiles of various transcription-associated factors around pseudogene loci. By integrating ∼18 000 RNA-seq data, we analysed the expression profiles of pseudogenes and explored their co-expression patterns with their parent genes in 32 cancers and 31 normal tissues. By combining microRNA binding sites, we demonstrated complex post-transcriptional regulation networks involving 275 microRNAs and 1201 pseudogenes. We generated ceRNA networks to illustrate the crosstalk between pseudogenes and their parent genes through competitive binding of microRNAs. In addition, we studied transcriptome-wide interactions between RNA binding proteins (RBPs) and pseudogenes based on 458 CLIP-seq datasets. In conjunction with epitranscriptome sequencing data, we also mapped 1039 RNA modification sites onto 635 pseudogenes. This database will provide insights into the transcriptional regulation, expression, functions and mechanisms of pseudogenes as well as their roles in biological processes and diseases.

Controlled releasing of regulations remains the most convenient method to deliver various drugs. In the present study, we precipitated gold nanoparticles with chrysophanol. The gold-chrysophanol into poly (DL-lactide-co-glycolide) nanoparticles was loaded and the biological activity of chrysophanol nanoparticles on human LNCap prostate cancer cells, was tested to acquire the sustained releasing property. The circular dichroism spectroscopy indicated that chrysophanol nanoparticles effectively resulted in conformational alterations in DNA and regulated different proteins associated with cell cycle arrest. The reactive oxygen species (ROS), apoptosis, cell cycle, DNA damage, Cyto-c and caspase-3 activity were analyzed, and the expression levels of different anti- and pro-apoptotic were studied using immunoblotting analysis. The cytotoxicity assay suggested that chrysophanol nanoparticles preferentially killed prostate cancer cells in comparison to the normal cells. Chrysophanol nanoparticles reduced histone deacetylases (HDACs) to suppress cell proliferation and induce apoptosis by arresting the cell cycle in sub-G phase. In addition, the cell cycle-related proteins, including p27, CHK1, cyclin D1, CDK1, p-AMP-activated protein kinase (AMPK) and p-protein kinase B (AKT), were regulated by chrysophanol nanoparticles to prevent human prostate cancer cell progression. Chrysophanol nanoparticles induced apoptosis in LNCap cells by promoting p53/ROS crosstalk to prevent proliferation. Pharmacokinetic study in mice indicated that chrysophanol nanoparticle injection showed high bioavailability compared to the free chrysophanol. Also, in vivo study revealed that chrysophanol nanoparticles obviously reduced tumor volume and weight. In conclusion, the data above suggested that chrysophanol nanoparticles might be effective to prevent human prostate cancer progression.

LTR retrotransposons are abundant repetitive elements in the human genome, but their functions remain poorly understood. Here, we report the function and regulatory mechanism of an ERV-9 LTR retrotransposon-derived lncRNA called p53-regulated lncRNA for homologous recombination (HR) repair 1 (PRLH1) in human cells. PRLH1 is highly expressed in p53-mutated hepatocellular carcinoma (HCC) samples and promotes cell proliferation in p53-mutated HCC cells, and its transcription is promoted by NF-Y and suppressed by p53. Mechanistically, PRLH1 specifically binds to an uncharacterized domain of RNF169 through two GCUUCA boxes in its 5' terminal region to form a DNA repair complex that supplants 53BP1 at double-strand break (DSB) sites and then promotes the initiation of HR repair. Notably, PRLH1 is essential for the stabilization of RNF169, acting as an RNA platform to recruit and assemble HR protein factors. This study characterizes PRLH1 as a novel HR-promoting factor and provides new insights into the function and mechanism of LTR retrotransposon-derived lncRNAs.

MicroRNA-122 (miR-122) is the most abundant microRNA in hepatocytes and a central player in liver biology and disease. Herein, we report a previously unknown role for miR-122 in hepatocyte intrinsic innate immunity. Restoration of miR-122 levels in hepatoma cells markedly enhanced the activation of interferons (IFNs) in response to a variety of viral nucleic acids or simulations, especially in response to hepatitis C virus RNA and poly (I:C). Mechanistically, miR-122 downregulated the phosphorylation (Tyr705) of STAT3, thereby removing the negative regulation of STAT3 on IFN-signaling. STAT3 represses IFN expression by inhibiting interferon regulatory factor 1 (IRF1), whereas miR-122 targets MERTK, FGFR1 and IGF1R, three receptor tyrosine kinases (RTKs) that directly promote STAT3 phosphorylation. This work identifies a miR-122–RTKs/STAT3–IRF1–IFNs regulatory circuitry, which may play a pivotal role in regulating hepatocyte innate immunity. These findings renewed our knowledge of miR-122’s function and have important implications for the treatment of hepatitis viruses.

The abnormal transcriptional regulation of non-coding RNAs (ncRNAs) and protein-coding genes (PCGs) is contributed to various biological processes and linked with human diseases, but the underlying mechanisms remain elusive. In this study, we developed ChIPBase v2.0 (http://rna.sysu.edu.cn/chipbase/) to explore the transcriptional regulatory networks of ncRNAs and PCGs. ChIPBase v2.0 has been expanded with ∼10 200 curated ChIP-seq datasets, which represent about 20 times expansion when comparing to the previous released version. We identified thousands of binding motif matrices and their binding sites from ChIP-seq data of DNA-binding proteins and predicted millions of transcriptional regulatory relationships between transcription factors (TFs) and genes. We constructed 'Regulator' module to predict hundreds of TFs and histone modifications that were involved in or affected transcription of ncRNAs and PCGs. Moreover, we built a web-based tool, Co-Expression, to explore the co-expression patterns between DNA-binding proteins and various types of genes by integrating the gene expression profiles of ∼10 000 tumor samples and ∼9100 normal tissues and cell lines. ChIPBase also provides a ChIP-Function tool and a genome browser to predict functions of diverse genes and visualize various ChIP-seq data. This study will greatly expand our understanding of the transcriptional regulations of ncRNAs and PCGs.

// Yan-Hua Guo 1, 3, * , Lu-Qin Wang 1, * , Bin Li 1, * , Hui Xu 1 , Jian-Hua Yang 1 , Li-Si Zheng 1 , Peng Yu 1 , Ai-Dong Zhou 2 , Yin Zhang 1 , Shu-Juan Xie 1 , Zi-Rui Liang 1 , Chen-Min Zhang 1 , Hui Zhou 1, Liang-Hu Qu 1 1 Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou, P. R. China 2 Present address: Department of Neurosurgery, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA 3 Present address: Guangzhou Quality Supervision and Testing Institute, Guangzhou, China * These authors have contributed equally to this work Correspondence to: Liang-Hu Qu, email: lssqlh@mail.sysu.edu.cn Keywords: Wnt/β-catenin, miR-150, CREB, EMT, colorectal cancer Received: December 16, 2015 Accepted: May 09, 2016 Published: June 7, 2016 ABSTRACT A hallmark of aberrant activation of the Wnt/β-catenin signaling pathway has been observed in most colorectal cancers (CRC), but little is known about the role of non-coding RNAs regulated by this pathway. Here, we found that miR-150 was the most significantly upregulated microRNA responsive to elevated of Wnt/β-catenin signaling activity in both HCT116 and HEK293T cells. Mechanistically, the β-catenin/LEF1 complex binds to the conserved TCF/LEF1-binding element in the miR-150 promoter and thereby transactivates its expression. Enforced expression of miR-150 in HCT116 cell line transformed cells into a spindle shape with higher migration and invasion activity. miR-150 markedly suppressed the CREB signaling pathway by targeting its core transcription factors CREB1 and EP300. Knockdown of CREB1 or EP300 and knockout of CREB1 by CRISPR/Cas9 phenocopied the epithelial-mesenchymal transition (EMT) observed in HCT116 cells in response to miR-150 overexpression. In summary, our data indicate that miR-150 is a novel Wnt effector that may significantly enhance EMT of CRC cells by targeting the CREB signaling pathway.

tRNA-derived small RNA fragments (tRFs) are one class of small non-coding RNAs derived from transfer RNAs (tRNAs). tRFs play important roles in cellular processes and are involved in multiple cancers. High-throughput small RNA (sRNA) sequencing experiments can detect all the cellular expressed sRNAs, including tRFs. However, distinguishing genuine tRFs from RNA fragments generated by random degradation remains a major challenge. In this study, we developed an integrated web-based computing system, tRF2Cancer, to accurately identify tRFs from sRNA deep-sequencing data and evaluate their expression in multiple cancers. The binomial test was introduced to evaluate whether reads from a small RNA-seq data set represent tRFs or degraded fragments. A classification method was then used to annotate the types of tRFs based on their sites of origin in pre-tRNA or mature tRNA. We applied the pipeline to analyze 10 991 data sets from 32 types of cancers and identified thousands of expressed tRFs. A tool called 'tRFinCancer' was developed to facilitate the users to inspect the expression of tRFs across different types of cancers. Another tool called 'tRFBrowser' shows both the sites of origin and the distribution of chemical modification sites in tRFs on their source tRNA. The tRF2Cancer web server is available at http://rna.sysu.edu.cn/tRFfinder/.

// Ze-Lin Wang 1, 2, * , Bin Li 1, 2, * , Stephen R. Piccolo 3, 4 , Xiao-Qin Zhang 5 , Jun-Hao Li 1, 2 , Hui Zhou 1, 2 , Jian-Hua Yang 1, 2 , Liang-Hu Qu 1, 2 1 Key Laboratory of Gene Engineering of the Ministry of Education, Sun Yat-sen University, Guangzhou, China 2 State Key Laboratory for Biocontrol, Sun Yat-sen University, Guangzhou, China 3 Department of Biology, Brigham Young University, Provo, Utah, USA 4 Department of Biomedical Informatics, University of Utah, Salt Lake City, Utah, USA 5 Department of Surgery, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong * These authors have contributed equally to this work Correspondence to: Jian-Hua Yang, e-mail: yangjh7@mail.sysu.edu.cn Liang-Hu Qu, e-mail: lssqlh@mail.sysu.edu.cn Keywords: lncRNA, expression profile, prognostification, somatic copy number alteration Received: October 22, 2015 Accepted: April 11, 2016 Published: April 27, 2016 ABSTRACT Long non-coding RNAs (lncRNAs) have been shown to contribute to tumorigenesis. However, surprisingly little is known about the comprehensive clinical and genomic characterization of lncRNAs across human cancer. In this study, we conducted comprehensive analyses for the expression profile, clinical outcomes, somatic copy number alterations (SCNAs) profile of lncRNAs in ~7000 clinical samples from 15 different cancer types. We identified significantly differentially expressed lncRNAs between tumor and normal tissues from each cancer. Notably, we characterized 47 lncRNAs which were extensively dysregulated in at least 10 cancer types, suggesting a conserved function in cancer development. We also analyzed the associations between lncRNA expressions and patient survival, and identified sets of lncRNAs that possessed significant prognostic values in specific cancer types. Our combined analysis of SCNA data and expression data uncovered 116 dysregulated lncRNAs are strikingly genomic altered across 15 cancer types, indicating their oncogenic potentials. Our study may lay the groundwork for future functional studies of lncRNAs and help facilitate the discovery of novel clinical biomarkers.

The intrinsic innate immunity of hepatocytes is essential for the control of hepatitis viruses and influences the outcome of antiviral therapy. MicroRNA-122 (miR-122) is the most abundant microRNA in hepatocytes and is a central player in liver biology and disease. However, little is known about the role of miR-122 in hepatocyte innate immunity. Herein, we show that restoring miR-122 levels in hepatoma cells markedly increased the activation of both type III and type I interferons (IFNs) in response to hepatitis C virus (HCV) RNA or poly(I:C). We determined that miR-122 promotes IFN production through down-regulating the tyrosine (Tyr705) phosphorylation of STAT3. We show that STAT3 represses IFN activation by inhibiting interferon regulatory factor 1 (IRF1), which is rate-limiting for maximal IFN expression, especially type III IFNs. Through large-scale screening, we identified that miR-122 targets MERTK, FGFR1 and IGF1R, three oncogenic receptor tyrosine kinases that directly promote STAT3 phosphorylation. These findings reveal a previously unknown role for miR-122 in hepatic immunity and indicate a new potential strategy for treating hepatic infections through targeting STAT3.

Background: Alternations in mitochondrial DNA (mtDNA) have long been proposed to be involved in the pathogenesis of ovarian cancer (OC). However, the allele frequency of mitochondrial tRNA (mttRNA) variants in OC remains largely understood. Objective: To explore the potential association between mt-tRNA variants and OC. Methods: 95 OC patients and 50 control subjects were participated in this study. The 22 mt-tRNA genes were PCR amplified and subsequently sequenced by ABI-3700 automatic DNA sequencer. Moreover, we used phylogenetic approach to determine the pathogenic status of these mt-tRNA variants. Results: We identified 4 mt-tRNA variants: the tRNAThr A15951G; tRNAHis G12192A; tRNAGlu A14693G and tRNALeu(UUR) A3302G, whereas these tRNA variants were absent in healthy controls. Moreover, these variants localized at positions which were highly conserved between different species, and may cause the failure in mt-tRNAs metabolism, consequently result in mitochondrial dysfunction that responsible for OC. Conclusions: Variations in mt-tRNAs may be associated with OC.

Small non-coding RNAs (e.g. miRNAs) and long non-coding RNAs (e.g. lincRNAs and circRNAs) are emerging as key regulators of various cellular processes. However, only a very small fraction of these enigmatic RNAs have been well functionally characterized. In this study, we describe deepBase v2.0 (http://biocenter.sysu.edu.cn/deepBase/), an updated platform, to decode evolution, expression patterns and functions of diverse ncRNAs across 19 species. deepBase v2.0 has been updated to provide the most comprehensive collection of ncRNA-derived small RNAs generated from 588 sRNA-Seq datasets. Moreover, we developed a pipeline named lncSeeker to identify 176 680 high-confidence lncRNAs from 14 species. Temporal and spatial expression patterns of various ncRNAs were profiled. We identified approximately 24 280 primate-specific, 5193 rodent-specific lncRNAs, and 55 highly conserved lncRNA orthologs between human and zebrafish. We annotated 14 867 human circRNAs, 1260 of which are orthologous to mouse circRNAs. By combining expression profiles and functional genomic annotations, we developed lncFunction web-server to predict the function of lncRNAs based on protein-lncRNA co-expression networks. This study is expected to provide considerable resources to facilitate future experimental studies and to uncover ncRNA functions.

Although more than 100 different types of RNA modifications have been characterized across all living organisms, surprisingly little is known about the modified positions and their functions. Recently, various high-throughput modification sequencing methods have been developed to identify diverse post-transcriptional modifications of RNA molecules. In this study, we developed a novel resource, RMBase (RNA Modification Base, http://mirlab.sysu.edu.cn/rmbase/), to decode the genome-wide landscape of RNA modifications identified from high-throughput modification data generated by 18 independent studies. The current release of RMBase includes ∼ 9500 pseudouridine (Ψ) modifications generated from Pseudo-seq and CeU-seq sequencing data, ∼ 1000 5-methylcytosines (m(5)C) predicted from Aza-IP data, ∼ 124 200 N6-Methyladenosine (m(6)A) modifications discovered from m(6)A-seq and ∼ 1210 2'-O-methylations (2'-O-Me) identified from RiboMeth-seq data and public resources. Moreover, RMBase provides a comprehensive listing of other experimentally supported types of RNA modifications by integrating various resources. It provides web interfaces to show thousands of relationships between RNA modification sites and microRNA target sites. It can also be used to illustrate the disease-related SNPs residing in the modification sites/regions. RMBase provides a genome browser and a web-based modTool to query, annotate and visualize various RNA modifications. This database will help expand our understanding of potential functions of RNA modifications.

Marek׳s disease virus (MDV) is a representative alpha herpes virus able to induce rapid-onset T-cell lymphoma in its natural host and regarded as an ideal model for the study of virus-induced tumorigenesis. Recent studies have shown that the mdv1-miR-M4-5p, a viral analog of cellular miR-155, is critical for MDV׳s oncogenicity. However, the precise mechanism whereby it was involved in MD lymphomagenesis remained unknown. We have presently identified the host mRNA targets of mdv1-miR-M4-5 and identified the latent TGF-β binding protein 1 (LTBP1) as a critical target for it. We found that during MDV infection, down-regulation of LTBP1 expression by mdv1-miR-M4-5p led to a significant decrease of the secretion and activation of TGF-β1, with suppression of TGF-β signaling and a significant activation of expression of c-Myc, a well-known oncogene which is critical for virus-induced tumorigenesis. Our findings reveal a novel and important mechanism of how mdv1-miR-M4-5p potentially contributes to MDV-induced tumorigenesis.

microRNAs (miRNAs) are important nonprotein-coding genes that are involved in almost all biological processes, including cell differentiation and fate determination, developmental regulation, and immune responses. Investigations have shown that some miRNAs can highly affect plant agricultural traits, including virus resistance, nematode resistance, drought and salinity tolerance, heavy metal detoxification, biomass yield, grain yield, fruit development and flower development. Therefore, these miRNAs are considered a newly identified gene resource for the genetic improvement of crops. In this review, we will summarize the recent findings of the rice miRNA-directed regulatory network, which controls agronomic traits such as yield, quality and stress tolerance, and explore the outlook for the uses of these miRNA-associated traits in rice biotechnology.

The pervasive transcription of the genome creates many types of non-coding RNAs (ncRNAs). However, we know very little regarding the functions and the regulatory mechanisms of these ncRNAs. Exploring the interactions of RNA and RNA binding proteins (RBPs) is vital because it can allow us to truly understand how these ncRNAs behave in vivo. High-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP or CLIP-seq) and its variants have been successfully used as systemic techniques to study RBP binding sites. In this review, we will explain the major differences between the CLIP techniques, summarize successful applications of these techniques, discuss limitations of CLIP, present some suggested solutions and project their promising future roles in studying the RNA world.

Skeletal muscle differentiation is controlled by multiple cell signaling pathways, however, the JNK/MAPK signaling pathway dominating this process has not been fully elucidated. Here, we report that the JNK/MAPK pathway was significantly downregulated in the late stages of myogenesis, and in contrast to P38/MAPK pathway, it negatively regulated skeletal muscle differentiation. Based on the PAR-CLIP-seq analysis, we identified six elevated miRNAs (miR-1a-3p, miR-133a-3p, miR-133b-3p, miR-206-3p, miR-128-3p, miR-351-5p), namely myogenesis-associated miRNAs (mamiRs), negatively controlled the JNK/MAPK pathway by repressing multiple factors for the phosphorylation of the JNK/MAPK pathway, including MEKK1, MEKK2, MKK7, and c-Jun but not JNK protein itself, and as a result, expression of transcriptional factor MyoD and mamiRs were further promoted. Our study revealed a novel double-negative feedback regulatory pattern of cell-specific miRNAs by targeting phosphorylation kinase signaling cascade responsible for skeletal muscle development.

Although tens of thousands of circular RNAs (circRNAs) have been identified in mammalian genomes, only few of them have been characterized with biological functions. Here, we report a new approach, circScan, to identify regulatory interactions between circRNAs and RNA-binding proteins (RBPs) by discovering back-splicing reads from Cross-Linking and Immunoprecipitation followed by high-throughput sequencing (CLIP-seq) data. By using our method, we have systematically scanned ~1500 CLIP-seq datasets, and identified ~12540 and ~1090 novel circRNA-RBP interactions in human and mouse genomes, respectively, which include all known interactions between circRNAs and Argonaute (AGO) proteins. More than twenty novel interactions were further experimentally confirmed by RNA Immunoprecipitation quantitative PCR (RIP-qPCR). Importantly, we uncovered that some natural circRNAs interacted with cap-independent translation factors eukaryotic initiation factor 3 (eIF3) and N6-Methyladenosine (m6A), indicating they can be translated into proteins. These findings demonstrate that circRNAs are regulated by various RBPs, suggesting they may play important roles in diverse biological processes.

Inducing degradation of oncoproteins by small molecule compounds has the potential to avoid drug resistance and therefore deserves to be exploited for new therapies. Oridonin is a natural compound with promising antitumor efficacy that can trigger the degradation of oncoproteins; however, the direct cellular targets and underlying mechanisms remain unclear. Here we report that oridonin depletes BCR-ABL through chaperon-mediated proteasomal degradation in leukemia. Mechanistically, oridonin poses oxidative stress in cancer cells and directly binds to cysteines of HSF1, leading to the activation of this master regulator of the chaperone system. The resulting induction of HSP70 and ubiquitin proteins and the enhanced binding to CHIP E3 ligase hence target BCR-ABL for ubiquitin-proteasome degradation. Both wild-type and mutant forms of BCR-ABL can be efficiently degraded by oridonin, supporting its efficacy observed in cultured cells as well as mouse tumor xenograft assays with either imatinib-sensitive or -resistant cells. Collectively, our results identify a novel mechanism by which oridonin induces rapid degradation of BCR-ABL as well as a novel pharmaceutical activator of HSF1 that represents a promising treatment for leukemia.

The onset of cancer is a complex process that is driven by the accumulation of multiple genetic mutations. However, the fact that inhibition of a single oncogene can impair the proliferation and survival of cancer cells due to their "oncogene addiction" provides implications for the so-called "molecular targeted therapy" in cancer treatment. The oncogenic transcription factor c-Myc is overexpressed in many types of cancers, and as a typical oncogene to which many cancers are addicted, c-Myc is necessary for the rapid proliferation of cancer cells. Strategies aimed at targeting c-Myc, including interfering with c-Myc synthesis, stability and transcriptional activity, have emerged as effective cancer treatments. We have recently shown that a natural agent, oridonin, promotes the Fbw7-mediated proteasomal degradation of c-Myc, leading to subsequent cell growth inhibition and apoptosis and demonstrating a new c-Myc-targeting strategy. Despite the effectiveness of molecular targeting in cancer treatment, failure to achieve long-lasting efficacy with a single agent is observed because cancer cells can recover from oncogene addiction as a result of their genomic instability and heterogeneity. Combined cancer therapies were therefore developed and showed better efficacies than single-agent therapy in cancer cell lines and mouse models. Combined therapy based on c-Myc targeting can be achieved through various strategies. Agents that also target c-Myc but use different mechanisms, or agents that act on other genes in the c-Myc pathway, can be selected for combination. In addition, the targeting of genes involved in different cellular processes in other pathways might also be a successful strategy. Regardless of the therapy adopted, it is important to first determine the molecular mechanisms underlying the agents to inform the therapy design. Among the various targets of therapeutic agents is a family of noncoding small RNAs, called microRNAs, that have been implicated in the anti-cancer activity of many therapeutic agents. c-Myc, as a transcription factor, regulates the expression of many microRNAs and is in turn regulated by microRNAs. Combining c-Myc-targeting agents with those that target microRNAs might provide a novel approach for cancer therapy.

Coordinated assembly of the ribosome is essential for proper translational activity in eukaryotic cells. It is therefore critical to coordinate the expression of components of ribosomal programs with the cell's nutritional status. However, coordinating expression of these components is poorly understood. Here, by combining experimental and computational approaches, we systematically identified box C/D snoRNAs in four fission yeasts and found that the expression of box C/D snoRNA and ribosomal protein (RP) genes were orchestrated by a common Homol-D box, thereby ensuring a constant balance of these two genetic components. Interestingly, such transcriptional coregulations could be observed in most Ascomycota species and were mediated by different cis-regulatory elements. Via the reservation of cis elements, changes in spatial configuration, the substitution of cis elements, and gain or loss of cis elements, the regulatory networks of box C/D snoRNAs evolved to correspond with those of the RP genes, maintaining transcriptional coregulation between box C/D snoRNAs and RP genes. Our results indicate that coregulation via common cis elements is an important mechanism to coordinate expression of the RP and snoRNA genes, which ensures a constant balance of these two components.

Plant laccase (LAC) enzymes belong to the blue copper oxidase family and polymerize monolignols into lignin. Recent studies have established the involvement of microRNAs in this process; however, physiological functions and regulation of plant laccases remain poorly understood. Here, we show that a laccase gene, LAC4, regulated by a microRNA, miR397b, controls both lignin biosynthesis and seed yield in Arabidopsis. In transgenic plants, overexpression of miR397b (OXmiR397b) reduced lignin deposition. The secondary wall thickness of vessels and the fibres was reduced in the OXmiR397b line, and both syringyl and guaiacyl subunits are decreased, leading to weakening of vascular tissues. In contrast, overexpression of miR397b-resistant laccase mRNA results in an opposite phenotype. Plants overexpressing miR397b develop more than two inflorescence shoots and have an increased silique number and silique length, resulting in higher seed numbers. In addition, enlarged seeds and more seeds are formed in these miR397b overexpression plants. The study suggests that miR397-mediated development via regulating laccase genes might be a common mechanism in flowering plants and that the modulation of laccase by miR397 may be potential for engineering plant biomass production with less lignin.

The microRNA (miRNA) mdv1-miR-M4, a functional miR-155 ortholog encoded by oncogenic Marek's disease virus (MDV), has previously been suggested to be involved in MDV pathogenesis. Using the technique of bacterial artificial chromosome mutagenesis, we have presently evaluated the potential role of mdv1-miR-M4 in the oncogenesis of the very virulent (vv) MDV strain GX0101. Unexpectedly, deletions of the Meq-cluster or mdv1-miR-M4 alone from the viral genome strongly decreased rather than abolished its oncogenicity. Compared to GX0101, mortalities of mutants GXΔmiR-M4 and GXΔMeq-miRs were reduced from 100% to 18% and 4%, coupled with the gross tumor incidence reduction from 28% to 22% and 8%, respectively. Our data suggests that the mdv1-miR-M4 is possibly an important regulator in the development of Marek's disease (MD) lymphomas but is not essential for the oncogenicity of vvMDV. In addition, some of the other Meq-clustered miRNAs may also play potentially critical roles in vvMDV induction of lymphomas.

Although microRNAs (miRNAs), other non-coding RNAs (ncRNAs) (e.g. lncRNAs, pseudogenes and circRNAs) and competing endogenous RNAs (ceRNAs) have been implicated in cell-fate determination and in various human diseases, surprisingly little is known about the regulatory interaction networks among the multiple classes of RNAs. In this study, we developed starBase v2.0 (http://starbase.sysu.edu.cn/) to systematically identify the RNA-RNA and protein-RNA interaction networks from 108 CLIP-Seq (PAR-CLIP, HITS-CLIP, iCLIP, CLASH) data sets generated by 37 independent studies. By analyzing millions of RNA-binding protein binding sites, we identified ∼9000 miRNA-circRNA, 16 000 miRNA-pseudogene and 285,000 protein-RNA regulatory relationships. Moreover, starBase v2.0 has been updated to provide the most comprehensive CLIP-Seq experimentally supported miRNA-mRNA and miRNA-lncRNA interaction networks to date. We identified ∼10,000 ceRNA pairs from CLIP-supported miRNA target sites. By combining 13 functional genomic annotations, we developed miRFunction and ceRNAFunction web servers to predict the function of miRNAs and other ncRNAs from the miRNA-mediated regulatory networks. Finally, we developed interactive web implementations to provide visualization, analysis and downloading of the aforementioned large-scale data sets. This study will greatly expand our understanding of ncRNA functions and their coordinated regulatory networks.

MicroRNAs (miRNAs) are a class of non-coding small RNAs that consist of ≈ 22 nt and are involved in several biological processes by regulating target gene expression. MiR-138 has many biological functions and is often downregulated in cancers. Our results showed that overexpression of miR-138 downregulated target RMND5A (required for meiotic nuclear division 5 homolog A) and reduced Exportin-5 stability, which results in decreased levels of pre-miRNA nuclear export in HeLa cells. We also found that miR-138 could significantly inhibit HeLa cell migration by targeting RMND5A. Our study therefore identifies miR-138-RMND5A-Exportin-5 as a previously unknown miRNA processing regulatory pathway in HeLa cells.