Glycolysis and tumor progression promoted by the m6A writer VIRMA via m6A-dependent upregulation of STRA6 in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies, highlighting the urgent need to elucidate the underlying oncogenic mechanisms. VIRMA is a classic isoform of methyltransferases that participates in epigenetic transcriptomic modification in eukaryotic mRNAs. However, the exact roles of VIRMA in PDAC remain unclear. Here, we identified that VIRMA is highly expressed in PDAC, and histone modifications of the promoter may partly account for this dysregulation. Moreover, VIRMA is closely related to glycolysis and poor prognosis in PDAC. We further determined that STRA6 is a direct downstream target of VIRMA in PDAC by RNA sequencing (RNA-seq) and m6A sequencing (m6A-seq). VIRMA is involved in gene expression regulation via 3' UTR targeting of STRA6 mRNA. Furthermore, the m6A reader IGF2BP2 was shown to critically contribute to the stability of STRA6 mRNA. We describe the role of VIRMA in promoting signaling via the STRA6/STAT3 axis, which results in increased levels of HIF-1α, a key activator of glycolysis. In vivo and in vitro experiments reveal that the VIRMA-STRA6-STAT3-HIF-1α axis plays an instrumental role in glycolysis and tumor progression in PDAC. In conclusion, we demonstrate that VIRMA can increase glycolysis in PDAC by upregulating STRA6, a cell surface membrane protein that stimulates the STAT3 pathway, thereby activating HIF-1α and leading to pancreatic cancer malignancy. Overall, our data strongly suggest that the VIRMA-STRA6-STAT3-HIF-1α axis is a viable therapeutic target in PDAC.

Noncoding RNAs in skeletal development and disorders

Protein-encoding genes only constitute less than 2% of total human genomic sequences, and 98% of genetic information was previously referred to as "junk DNA". Meanwhile, non-coding RNAs (ncRNAs) consist of approximately 60% of the transcriptional output of human cells. Thousands of ncRNAs have been identified in recent decades, and their essential roles in the regulation of gene expression in diverse cellular pathways associated with fundamental cell processes, including proliferation, differentiation, apoptosis, and metabolism, have been extensively investigated. Furthermore, the gene regulation networks they form modulate gene expression in normal development and under pathological conditions. In this review, we integrate current information about the classification, biogenesis, and function of ncRNAs and how these ncRNAs support skeletal development through their regulation of critical genes and signaling pathways in vivo. We also summarize the updated knowledge of ncRNAs involved in common skeletal diseases and disorders, including but not limited to osteoporosis, osteoarthritis, rheumatoid arthritis, scoliosis, and intervertebral disc degeneration, by highlighting their roles established from in vivo, in vitro, and ex vivo studies.

Niche stiffness sustains cancer stemness via TAZ and NANOG phase separation.

Emerging evidence shows that the biomechanical environment is required to support cancer stem cells (CSCs), which play a crucial role in drug resistance. However, how mechanotransduction signals regulate CSCs and its clinical significance has remained unclear. Using clinical-practice ultrasound elastography for patients' lesions and atomic force microscopy for surgical samples, we reveal that increased matrix stiffness is associated with poor responses to neoadjuvant chemotherapy, worse prognosis, and CSC enrichment in patients with breast cancer. Mechanically, TAZ activated by biomechanics enhances CSC properties via phase separation with NANOG. TAZ-NANOG phase separation, which is dependent on acidic residues in the N-terminal activation domain of NANOG, promotes the transcription of SOX2 and OCT4. Therapeutically, targeting NANOG or TAZ reduces CSCs and enhances the chemosensitivity in vivo. Collectively, this study demonstrated that the phase separation of a pluripotency transcription factor links mechanical cues in the niche to the fate of CSCs.

Reprogramming of m6A epitranscriptome is crucial for shaping of transcriptome and proteome in response to hypoxia.

Hypoxia causes a series of responses supporting cells to survive in harsh environments. Substantial post-transcriptional and translational regulation during hypoxia has been observed. However, detailed regulatory mechanism in response to hypoxia is still far from complete. RNA m6A modification has been proven to govern the life cycle of RNAs. Here, we reported that total m6A level of mRNAs was decreased during hypoxia, which might be mediated by the induction of m6A eraser, ALKBH5. Meanwhile, expression levels of most YTH family members of m6A readers were systematically down-regulated. Transcriptome-wide analysis of m6A revealed a drastic reprogramming of m6A epitranscriptome during cellular hypoxia. Integration of m6A epitranscriptome with either RNA-seq based transcriptome analysis or mass spectrometry (LC-MS/MS) based proteome analysis of cells upon hypoxic stress revealed that reprogramming of m6A epitranscriptome reshaped the transcriptome and proteome, thereby supporting efficient generation of energy for adaption to hypoxia. Moreover, ATP production was blocked when silencing an m6A eraser, ALKBH5, under hypoxic condition, demonstrating that m6A pathway is an important regulator during hypoxic response. Collectively, our studies indicate that crosstalk between m6A and HIF1 pathway is essential for cellular response to hypoxia, providing insights into the underlying molecular mechanisms during hypoxia.

Protocol for Single-Cell Analysis of Tumor-Infiltrating B Cells Isolated from Human Breast Cancer Tissue Before and After Neo-adjuvant Chemotherapy.

Single-cell analysis of tumor-infiltrating lymphocytes obtained before and after preoperative therapy reflects the dynamic interplay of the tumor and immune system during treatment. Here, we present a protocol to implement single-cell analysis of tumor-infiltrating B cells, which were isolated from paired human breast cancers before and after neo-adjuvant chemotherapy. This protocol also facilitates isolation and single-cell analysis of other tumor-infiltrating lymphocytes. For complete information on the generation and use of this protocol, please refer to Lu et al. (2020).

ATM-Dependent Recruitment of BRD7 is required for Transcriptional Repression and DNA Repair at DNA Breaks Flanking Transcriptional Active Regions.

Repair of DNA double-strand breaks (DSBs) is essential for genome integrity, and is accompanied by transcriptional repression at the DSB regions. However, the mechanisms how DNA repair induces transcriptional inhibition remain elusive. Here, it is identified that BRD7 participates in DNA damage response (DDR) and is recruited to the damaged chromatin via ATM signaling. Mechanistically, BRD7 joins the polycomb repressive complex 2 (PRC2), the nucleosome remodeling and histone deacetylation (NuRD) complex at the damaged DNA and recruits E3 ubiquitin ligase RNF168 to the DSBs. Furthermore, ATM-mediated BRD7 phosphorylation is required for recruitment of the PRC2 complex, NuRD complex, DSB sensor complex MRE11-RAD50-NBS1 (MRN), and RNF168 to the active transcription sites at DSBs, resulting in transcriptional repression and DNA repair. Moreover, BRD7 deficiency sensitizes cancer cells to PARP inhibition. Collectively, BRD7 is crucial for DNA repair and DDR-mediated transcription repression, which may serve as a therapeutic target. The findings identify the missing link between DNA repair and transcription regulation that maintains genome integrity.

LncRNA DSCAM-AS1 interacts with YBX1 to promote cancer progression by forming a positive feedback loop that activates FOXA1 transcription network.

Rationale: The forkhead box A1 (FOXA1) is a crucial transcription factor in initiation and development of breast, lung and prostate cancer. Previous studies about the FOXA1 transcriptional network were mainly focused on protein-coding genes. Its regulatory network of long non-coding RNAs (lncRNAs) and their role in FOXA1 oncogenic activity remains unknown. Methods: The Cancer Genome Atlas (TCGA) data, RNA-seq and ChIP-seq data were used to analyze FOXA1 regulated lncRNAs. RT-qPCR was used to detect the expression of DSCAM-AS1, RT-qPCR and Western blotting were used to determine the expression of FOXA1, estrogen receptor α (ERα) and Y box binding protein 1 (YBX1). RNA pull-down and RIP-qPCR were employed to investigate the interaction between DSCAM-AS1 and YBX1. The effect of DSCAM-AS1 on malignant phenotypes was examined through in vitro and in vivo assays. Results: In this study, we conducted a global analysis of FOXA1 regulated lncRNAs. For detailed analysis, we chose lncRNA DSCAM-AS1, which is specifically expressed in lung adenocarcinoma, breast and prostate cancer. The expression level of DSCAM-AS1 is regulated by two super-enhancers (SEs) driven by FOXA1. High expression levels of DSCAM-AS1 was associated with poor prognosis. Knockout experiments showed DSCAM-AS1 was essential for the growth of xenograft tumors. Moreover, we demonstrated DSCAM-AS1 can regulate the expression of the master transcriptional factor FOXA1. In breast cancer, DSCAM-AS1 was also found to regulate ERα. Mechanistically, DSCAM-AS1 interacts with YBX1 and influences the recruitment of YBX1 in the promoter regions of FOXA1 and ERα. Conclusion: Our study demonstrated that lncRNA DSCAM-AS1 was transcriptionally activated by super-enhancers driven by FOXA1 and exhibited lineage-specific expression pattern. DSCAM-AS1 can promote cancer progression by interacting with YBX1 and regulating expression of FOXA1 and ERα.

Complement Signals Determine Opposite Effects of B Cells in Chemotherapy-Induced Immunity.

Understanding molecular mechanisms that dictate B cell diversity is important for targeting B cells as anti-cancer treatment. Through the single-cell dissection of B cell heterogeneity in longitudinal samples of patients with breast cancer before and after neoadjuvant chemotherapy, we revealed that an ICOSL+ B cell subset emerges after chemotherapy. Using three immunocompetent mouse models, we recapitulated the subset switch of human tumor-infiltrating B cells during chemotherapy. By employing B-cell-specific deletion mice, we showed that ICOSL in B cells boosts anti-tumor immunity by enhancing the effector to regulatory T cell ratio. The signature of ICOSL+ B cells is imprinted by complement-CR2 signaling, which is triggered by immunogenic cell death. Moreover, we identified that CD55, a complement inhibitory protein, determines the opposite roles of B cells in chemotherapy. Collectively, we demonstrated a critical role of the B cell subset switch in chemotherapy response, which has implications in designing novel anti-cancer therapies. VIDEO ABSTRACT.

The crosstalk between RNA m6A epitranscriptome and TGFß signaling pathway contributes to the arrest of cell cycle.

TGFß signaling pathway is critical for the cell division, differentiation and apoptosis, the aberrant regulation of which will result in severe diseases including cancer. N6-methyl-adenosine (m6A) is one of the most abundant modifications on mRNA, it is unclear yet how m6A epitranscriptome response to stimulation of TGFß. Here, we found that cellular m6A level of RNA was elevated after TGFß treatment, which might be regulated by upregulation of WTAP and METTL3. MeRIP-Seq of mRNAs of MCF7 with or without treated by TGFß showed that mRNA with upregulated m6A modification level after TGFß treatment were enriched in TGFß signaling pathway. Phosphorylated level of SMAD2 or SMAD3 induced by TGFß was impaired when WTAP was silenced. Moreover, the m6A modification and mRNA level of JunB, which is known as a cell cycle inhibitor, both were increased after induction of TGFß and decreased after knockdown of WTAP. Intriguingly, growth inhibition caused by TGFß was rescued in WTAP-knockdown cells. Collectively, these results reveal the key role that m6A pathway playing in the cell cycle arrest induced by TGFß signaling, providing new mechanisms explanation for growth inhibition mediated by TGFß.

EuRBPDB: a comprehensive resource for annotation, functional and oncological investigation of eukaryotic RNA binding proteins (RBPs).

RNA binding proteins (RBPs) are a large protein family that plays important roles at almost all levels of gene regulation through interacting with RNAs, and contributes to numerous biological processes. However, the complete list of eukaryotic RBPs including human is still unavailable. Here, we systematically identified RBPs in 162 eukaryotic species based on both computational analysis of RNA binding domains (RBDs) and large-scale RNA binding proteomic data, and established a comprehensive eukaryotic RBP database, EuRBPDB (http://EuRBPDB.syshospital.org). We identified a total of 311 571 RBPs with RBDs (corresponding to 6368 ortholog groups) and 3,651 non-canonical RBPs without known RBDs. EuRBPDB provides detailed annotations for each RBP, including basic information and functional annotation. Moreover, we systematically investigated RBPs in the context of cancer biology based on published literatures, PPI-network and large-scale omics data. To facilitate the exploration of the clinical relevance of RBPs, we additionally designed a cancer web interface to systematically and interactively display the biological features of RBPs in various types of cancers. EuRBPDB has a user-friendly web interface with browse and search functions, as well as data downloading function. We expect that EuRBPDB will be a widely-used resource and platform for both the communities of RNA biology and cancer biology.

CIRDES: an efficient genome-wide method for in vivo RNA-RNA interactome analysis.

Complex RNA-RNA interactions underlie fundamental biological processes. However, a large number of RNA-RNA interactions remain unknown. Most existing methods used to map RNA-RNA interactions are based on proximity ligation, but these strategies also capture a huge amount of intramolecular RNA secondary structures, making it almost impossible to detect most RNA-RNA interactions. To overcome this limitation, we developed an efficient, genome-wide method, Capture Interacting RNA and Deep Sequencing (CIRDES) for in vivo capturing of the RNA interactome. We designed multiple 20-nt CIRDES probes tiling the whole RNA sequence of interest. This strategy obtained high selectivity and low background noise proved by qRT-PCR data. CIRDES enriched target RNA and its interacting RNAs from cells crosslinked by formaldehyde in high efficiency. After hybridization and purification, the captured RNAs were converted to the cDNA library after a highly efficient ligation to a 3' end infrared-dye-conjugated RNA adapter based on adapter ligation library construction. Using CIRDES, we detected highly abundant known interacting RNA, as well as a large number of novel targets of U6 snRNA. The enrichment of U4 snRNA, which interacts with U6, confirmed the robustness of the identification of the RNA-RNA interaction by CIRDES. These results suggest that the CIRDES is an efficient strategy for genome-wide RNA-RNA interactome analysis.

The subunit of RNA N6-methyladenosine methyltransferase OsFIP regulates early degeneration of microspores in rice.

N6-Methyladenosine (m6A) RNA methylation plays important roles during development in different species. However, knowledge of m6A RNA methylation in monocots remains limited. In this study, we reported that OsFIP and OsMTA2 are the components of m6A RNA methyltransferase complex in rice and uncovered a previously unknown function of m6A RNA methylation in regulation of plant sporogenesis. Importantly, OsFIP is essential for rice male gametogenesis. Knocking out of OsFIP results in early degeneration of microspores at the vacuolated pollen stage and simultaneously causes abnormal meiosis in prophase I. We further analyzed the profile of rice m6A modification during sporogenesis in both WT and OsFIP loss-of-function plants, and identified a rice panicle specific m6A modification motif "UGWAMH". Interestingly, we found that OsFIP directly mediates the m6A methylation of a set of threonine protease and NTPase mRNAs and is essential for their expression and/or splicing, which in turn regulates the progress of sporogenesis. Our findings revealed for the first time that OsFIP plays an indispensable role in plant early sporogenesis. This study also provides evidence for the different functions of the m6A RNA methyltransferase complex between rice and Arabidopsis.

Genome-wide study of ER-regulated lncRNAs shows AP000439.3 may function as a key regulator of cell cycle in breast cancer.

Estrogen receptor (ER) plays important roles in cell growth, development and tumorigenesis. Although ER-regulated genes have been extensively investigated, little is known about roles of ER-regulated lncRNAs in breast cancer. Here, we conducted genome-wide study of ER-regulated lncRNAs by using RNA-seq, ChIP-seq and TCGA data. A total of identified 114 ER-regulated lncRNAs were identified, many of them were overexpressed in ER+ breast cancer and co-expressed with some key regulators. Silencing one of most prominent lncRNA, AP000439.3, resulted in inhibition of cell cycle progression and proliferation. Further study revealed AP000439.3 can regulate expression of CCND1 through enhancing estrogen receptor induction of CCND1. This finding revealed lncRNAs may serve as important effectors of ER in regulation of gene expression and cell phenotype in breast cancer.

Deep sequencing reveals a global reprogramming of lncRNA transcriptome during EMT.

Several studies have shown that long non-coding RNAs (lncRNAs) may play an essential role in Epithelial-Mesenchymal Transition (EMT), which is an important step in tumor metastasis; however, little is known about the global change of lncRNA transcriptome during EMT. To investigate how lncRNA transcriptome alterations contribute to EMT progression regulation, we deep-sequenced the whole-transcriptome of MCF10A as the cells underwent TGF-ß-induced EMT. RESULTS: Deep-sequencing results showed that the long RNA transcriptome of MCF10A had undergone global changes as early as 8h after treatment with TGF-ß. The expression of 3403 known and novel lncRNAs, and 570 known and novel circRNAs were altered during EMT. To identify the key lncRNA-regulator, we constructed the co-expression network and found all junction nodes in the network are lncRNAs. One junction node, RP6-65G23.5, was further verified as a key regulator of EMT. Intriguingly, we identified 216 clusters containing lncRNAs which were located in "gene desert" regions. The expressions of all lncRNAs in these clusters changed concurrently during EMT, strongly suggesting that these clusters might play important roles in EMT. Our study reveals a global reprogramming of lncRNAs transcriptome during EMT and provides clues for the future study of the molecular mechanism of EMT.

miR-142-5p and miR-130a-3p are regulated by IL-4 and IL-13 and control profibrogenic macrophage program.

Macrophages play a pivotal role in tissue fibrogenesis, which underlies the pathogenesis of many end-stage chronic inflammatory diseases. MicroRNAs are key regulators of immune cell functions, but their roles in macrophage's fibrogenesis have not been characterized. Here we show that IL-4 and IL-13 induce miR-142-5p and downregulate miR-130a-3p in macrophages; these changes sustain the profibrogenic effect of macrophages. In vitro, miR-142-5p mimic prolongs STAT6 phosphorylation by targeting its negative regulator, SOCS1. Blocking miR-130a relieves its inhibition of PPARγ, which coordinates STAT6 signalling. In vivo, inhibiting miR-142-5p and increasing miR-130a-3p expression with locked nucleic acid-modified oligonucleotides inhibits CCL4-induced liver fibrosis and bleomycin-induced lung fibrosis in mice. Furthermore, macrophages from the tissue samples of patients with liver cirrhosis and idiopathic pulmonary fibrosis display increased miR-142-5p and decreased miR-130a-3p expression. Therefore, miR-142-5p and miR-130a-3p regulate macrophage profibrogenic gene expression in chronic inflammation.

miR-21 induces myofibroblast differentiation and promotes the malignant progression of breast phyllodes tumors.

Phyllodes tumors of breast, even histologically diagnosed as benign, can recur locally and have metastatic potential. Histologic markers only have limited value in predicting the clinical behavior of phyllodes tumors. It remains unknown what drives the malignant progression of phyllodes tumors. We found that the expression of myofibroblast markers, α-smooth muscle actin (α-SMA), fibroblast activation protein (FAP), and stromal cell-derived factor-1 (SDF-1), is progressively increased in the malignant progression of phyllodes tumors. Microarray showed that miR-21 was one of the most significantly upregulated microRNAs in malignant phyllodes tumors compared with benign phyllodes tumors. In addition, increased miR-21 expression was primarily localized to α-SMA-positive myofibroblasts. More importantly, α-SMA and miR-21 are independent predictors of recurrence and metastasis, with their predictive value of recurrence better than histologic grading. Furthermore, miR-21 mimics promoted, whereas miR-21 antisense oligos inhibited, the expression of α-SMA, FAP, and SDF-1, as well as the proliferation and invasion of primary stromal cells of phyllodes tumors. The ability of miR-21 to induce myofibroblast differentiation was mediated by its regulation on Smad7 and PTEN, which regulate the migration and proliferation, respectively. In breast phyllodes tumor xenografts, miR-21 accelerated tumor growth, induced myofibroblast differentiation, and promoted metastasis. This study suggests an important role of myofibroblast differentiation in the malignant progression of phyllodes tumors that is driven by increased miR-21.

Deep sequencing of human nuclear and cytoplasmic small RNAs reveals an unexpectedly complex subcellular distribution of miRNAs and tRNA 3' trailers.

BACKGROUND: MicroRNAs (miRNAs) are approximately 22-nt small non-coding regulatory RNAs that have generally been considered to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: To determine the number of miRNAs localized to the nucleus, we systematically investigated the subcellular distribution of small RNAs (sRNAs) by independent deep sequencing sequenced of the nuclear and cytoplasmic pools of 18- to 30-nucleotide sRNAs from human cells. We identified 339 nuclear and 324 cytoplasmic known miRNAs, 300 of which overlap, suggesting that the majority of miRNAs are imported into the nucleus. With the exception of a few miRNAs evidently enriched in the nuclear pool, such as the mir-29b, the ratio of miRNA abundances in the nuclear fraction versus in the cytoplasmic fraction vary to some extent. Moreover, our results revealed that a large number of tRNA 3' trailers are exported from the nucleus and accumulate in the cytoplasm. These tRNA 3' trailers accumulate in a variety of cell types, implying that the biogenesis of tRNA 3' trailers is conserved and that they have a potential functional role in vertebrate cells. CONCLUSION/SIGNIFICANCE: Our results provide the first comprehensive view of the subcellular distribution of diverse sRNAs and new insights into the roles of miRNAs and tRNA 3' trailers in the cell.

Stress-induced tRNA-derived RNAs: a novel class of small RNAs in the primitive eukaryote Giardia lamblia.

Giardia lamblia is an early diverging and evolutionarily successful protozoan as it can enter into a dormant cyst stage from a vegetative trophozoite. During dormant stage, its metabolic rate decreases dramatically. However, to date, the regulatory molecules participating in the initiation and maintenance of this process have not been fully investigated. In this study, we have identified a class of abundant small RNAs named sitRNAs, which are approximately 46 nucleotides in length and accumulate in G. lamblia encysting cultures. Remarkably, they are derived from the 3' portion of fully matured tRNAs by cleavage of the anticodon left arm, with the 3' terminal CCA triplex still connected. During differentiation, only a limited portion of mature tRNAs is cleaved, but this cleavage occurs almost in the entire tRNA family. sitRNAs begin to accumulate as early as 3 h after initiation of encystation and are maintained at a relatively stable level during the whole process, exhibiting an expression peak at around 24 hr. Our studies further show that sitRNAs can be induced by several other stress factors, and in the case of serum deprivation, both tRNAs and sitRNAs degrade rapidly, with the accumulation of tRNA being halved. Our results may provide new insight into a novel mechanism for stressed G. lamblia to regulate gene expression globally.