Deep sequencing of short capped RNAs reveals novel families of noncoding RNAs.

In eukaryotes, capped RNAs include long transcripts such as messenger RNAs and long noncoding RNAs, as well as shorter transcripts such as spliceosomal RNAs, small nucleolar RNAs, and enhancer RNAs. Long capped transcripts can be profiled using cap analysis gene expression (CAGE) sequencing and other methods. Here, we describe a sequencing library preparation protocol for short capped RNAs, apply it to a differentiation time course of the human cell line THP-1, and systematically compare the landscape of short capped RNAs to that of long capped RNAs. Transcription initiation peaks associated with genes in the sense direction have a strong preference to produce either long or short capped RNAs, with one out of six peaks detected in the short capped RNA libraries only. Gene-associated short capped RNAs have highly specific 3' ends, typically overlapping splice sites. Enhancers also preferentially generate either short or long capped RNAs, with 10% of enhancers observed in the short capped RNA libraries only. Enhancers producing either short or long capped RNAs show enrichment for GWAS-associated disease SNPs. We conclude that deep sequencing of short capped RNAs reveals new families of noncoding RNAs and elucidates the diversity of transcripts generated at known and novel promoters and enhancers.

Inhibition of BCL2A1 by STAT5 inactivation overcomes resistance to targeted therapies of FLT3-ITD/D835 mutant AML.

Tyrosine kinase inhibitors (TKIs) are established drugs in the therapy of FLT3-ITD mutated acute myeloid leukemia (AML). However, acquired mutations, such as D835 in the tyrosine kinase domain (FLT3-ITD/D835), can induce resistance to TKIs. A cap analysis gene expression (CAGE) technology revealed that the gene expression of BCL2A1 transcription start sites was increased in primary AML cells bearing FLT3-ITD/D835 compared to FLT3-ITD. Overexpression of BCL2A1 attenuated the sensitivity to quizartinib, a type II TKI, and venetoclax, a selective BCL2 inhibitor, in AML cell lines. However, a type I TKI, gilteritinib, inhibited the expression of BCL2A1 through inactivation of STAT5 and alleviated TKI resistance of FLT3-ITD/D835. The combination of gilteritinib and venetoclax showed synergistic effects in the FLT3-ITD/D835 positive AML cells. The promoter region of BCL2A1 contains a BRD4 binding site. Thus, the blockade of BRD4 with a BET inhibitor (CPI-0610) downregulated BCL2A1 in FLT3-mutated AML cells and extended profound suppression of FLT3-ITD/D835 mutant cells. Therefore, we propose that BCL2A1 has the potential to be a novel therapeutic target in treating FLT3-ITD/D835 mutated AML.

Two ovarian candidate enhancers, identified by time series enhancer RNA analyses, harbor rare genetic variations identified in ovarian insufficiency.

The genetic regulation of ovarian development remains largely unclear. Indeed, in most cases of impaired ovarian development-such as 46,XX disorders of sex development (DSD) without SRY, and premature ovarian insufficiency (POI)-the genetic causes have not been identified, and the vast majority of disease-associated sequence variants could lie within non-coding regulatory sequences. In this study, we aimed to identify enhancers of five ovarian genes known to play key roles in early ovarian development, basing our analysis on the expression of enhancer derived transcripts (eRNAs), which are considered to characterize active enhancers. Temporal expression profile changes in mouse WT1-positive ovarian cells were obtained from cap analysis of gene expression at E13.5, E16.5 and P0. We compared the chronological expression profiles of ovarian-specific eRNA with expression profiles for each of the ovarian-specific genes, yielding two candidate sequences for enhancers of Wnt4 and Rspo1. Both sequences are conserved between mouse and human, and we confirmed their enhancer activities using transient expression assays in murine granulosa cells. Furthermore, by sequencing the region in patients with impaired ovarian development in 24 patients, such as POI, gonadal dysgenesis and 46,XX DSD, we identified rare single nucleotide variants in both sequences. Our results demonstrate that combined analysis of the temporal expression profiles of eRNA and mRNA of target genes presents a powerful tool for locating cis-element enhancers, and a means of identifying disease-associated sequence variants that lie within non-coding regulatory sequences, thus advancing an important unmet need in forward human genetics.

Isolation of SARS-CoV-2 from COVID-19 Patients and an Asymptomatic Individual.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China, in December 2019. Despite the recent introduction of vaccines against SARS-CoV-2, more effective vaccines and antiviral drugs must be developed. Here, we isolated five SARS-CoV-2 strains from four patients with coronavirus disease (COVID-19) and an asymptomatic individual using pharyngeal swabs, nasopharyngeal swabs, and sputum samples. Cytopathic effects in inoculated Vero cells were observed between days 3 and 7. SARS-CoV-2 infection was confirmed by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) and next-generation sequencing. Phylogenetic analyses of the whole genome sequences showed that the virus isolates from the clinical samples belonged to the Wuhan and European lineages. These findings and the isolated viruses may contribute to the development of diagnostic tools, vaccines, and antiviral drugs for COVID-19.

Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network.

Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism.

Nanopore sequencing reveals TACC2 locus complexity and diversity of isoforms transcribed from an intronic promoter.

Gene expression is controlled at the transcriptional and post-transcriptional levels. The TACC2 gene was known to be associated with tumors but the control of its expression is unclear. We have reported that activity of the intronic promoter p10 of TACC2 in primary lesion of endometrial cancer is indicative of lymph node metastasis among a low-risk patient group. Here, we analyze the intronic promoter derived isoforms in JHUEM-1 endometrial cancer cells, and primary tissues of endometrial cancers and normal endometrium. Full-length cDNA amplicons are produced by long-range PCR and subjected to nanopore sequencing followed by computational error correction. We identify 16 stable, 4 variable, and 9 rare exons including 3 novel exons validated independently. All variable and rare exons reside N-terminally of the TACC domain and contribute to isoform variety. We found 240 isoforms as high-confidence, supported by more than 20 reads. The large number of isoforms produced from one minor promoter indicates the post-transcriptional complexity coupled with transcription at the TACC2 locus in cancer and normal cells.

Spliceostatin A interaction with SF3B limits U1 snRNP availability and causes premature cleavage and polyadenylation.

RNA splicing, a highly conserved process in eukaryotic gene expression, is seen as a promising target for anticancer agents. Splicing is associated with other RNA processing steps, such as transcription and nuclear export; however, our understanding of the interaction between splicing and other RNA regulatory mechanisms remains incomplete. Moreover, the impact of chemical splicing inhibition on long non-coding RNAs (lncRNAs) has been poorly understood. Here, we demonstrate that spliceostatin A (SSA), a chemical splicing modulator that binds to the SF3B subcomplex of the U2 small nuclear ribonucleoprotein particle (snRNP), limits U1 snRNP availability in splicing, resulting in premature cleavage and polyadenylation of MALAT1, a nuclear lncRNA, as well as protein-coding mRNAs. Therefore, truncated transcripts are exported into the cytoplasm and translated, resulting in aberrant protein products. Our work demonstrates that active recycling of the splicing machinery maintains homeostasis of RNA processing beyond intron excision.

Cancer-associated fibroblast migration in non-small cell lung cancers is modulated by increased integrin α11 expression.

Cancer-associated fibroblasts (CAFs) regulate cancer progression through the modulation of extracellular matrix (ECM) and cancer cell adhesion. While undergoing a series of phenotypic changes, CAFs control cancer-stroma interactions through integrin receptor signaling. Here, we isolated CAFs from patients with non-small-cell lung cancer (NSCLC) and examined their gene expression profiles. We identified collagen type XI α1 (COL11A1), integrin α11 (ITGA11), and the ITGA11 major ligand collagen type I α1 (COL1A1) among the 390 genes that were significantly enriched in NSCLC-associated CAFs. Increased ITGA11 expression in cancer stroma was correlated with a poor clinical outcome in patients with NSCLC. Increased expression of fibronectin and collagen type I induced ITGA11 expression in CAFs. The cellular migration of CAFs toward collagen type I and fibronectin was promoted via ERK1/2 signaling, independently of the fibronectin receptor integrin α5ß1. Additionally, ERK1/2 signaling induced ITGA11 and COL11A1 expression in cancer stroma. We, therefore, propose that targeting ITGA11 and COL11A1 expressing CAFs to block cancer-stroma interactions may serve as a novel, promising anti-tumor strategy.

Significance of HMGA2 expression as independent poor prognostic marker in perihilar and distal cholangiocarcinoma resected with curative intent.

BACKGROUND: Extrahepatic cholangiocarcinoma requires invasive surgery and is associated with poor prognosis; thus, a prognostic biomarker is highly needed. Extrahepatic cholangiocarcinoma is sub-classified into two types based on their location, namely perihilar and distal. Perihilar cholangiocarcinoma requires lobectomy as curative surgical resection, whereas the distal requires pancreatoduodenectomy. HMGA2 overexpression is reported to correlate with progression, aggressiveness, dissemination and poor prognosis in several types of cancers. Although its association with extrahepatic cholangiocarcinoma has been reported, none of the previous studies assessed its significance in each subtype. METHODS: We assessed the expression of HMGA2 protein in surgical specimens after curative intent surgery in 80 patients including 41 with perihilar cholangiocarcinoma and 39 with distal cholangiocarcinoma by immunohistochemistry. We then examined its association with clinicopathological findings and patient survival outcomes. RESULTS: We found that HMGA2 was expressed in 51% (21 of 41) of perihilar cholangiocarcinoma and 41% (16 of 39) of distal cholangiocarcinoma samples. In perihilar cholangiocarcinoma, we found significant correlations between expression and vascular invasion and perineural invasion. In distal cholangiocarcinoma, we found that protein levels correlated with tumor grade. Univariate and multivariate analyses demonstrated that HMGA2 expression was an independent poor prognostic factor for patients with both subtypes of disease. CONCLUSIONS: Our results revealed that HMGA2 expression as an independent prognostic marker for both perihilar and distal cholangiocarcinoma that were resected with curative intent.

FANTOM enters 20th year: expansion of transcriptomic atlases and functional annotation of non-coding RNAs.

The Functional ANnoTation Of the Mammalian genome (FANTOM) Consortium has continued to provide extensive resources in the pursuit of understanding the transcriptome, and transcriptional regulation, of mammalian genomes for the last 20 years. To share these resources with the research community, the FANTOM web-interfaces and databases are being regularly updated, enhanced and expanded with new data types. In recent years, the FANTOM Consortium's efforts have been mainly focused on creating new non-coding RNA datasets and resources. The existing FANTOM5 human and mouse miRNA atlas was supplemented with rat, dog, and chicken datasets. The sixth (latest) edition of the FANTOM project was launched to assess the function of human long non-coding RNAs (lncRNAs). From its creation until 2020, FANTOM6 has contributed to the research community a large dataset generated from the knock-down of 285 lncRNAs in human dermal fibroblasts; this is followed with extensive expression profiling and cellular phenotyping. Other updates to the FANTOM resource includes the reprocessing of the miRNA and promoter atlases of human, mouse and chicken with the latest reference genome assemblies. To facilitate the use and accessibility of all above resources we further enhanced FANTOM data viewers and web interfaces. The updated FANTOM web resource is publicly available at https://fantom.gsc.riken.jp/.

CREB3L1 overexpression as a potential diagnostic marker of Philadelphia chromosome-negative myeloproliferative neoplasms.

Discrimination of Philadelphia-negative myeloproliferative neoplasms (Ph-MPNs) from reactive hypercytosis and myelofibrosis requires a constellation of testing including driver mutation analysis and bone marrow biopsies. We searched for a biomarker that can more easily distinguish Ph-MPNs from reactive hypercytosis and myelofibrosis by using RNA-seq analysis utilizing platelet-rich plasma (PRP)-derived RNAs from patients with essential thrombocythemia (ET) and reactive thrombocytosis, and CREB3L1 was found to have an extremely high impact in discriminating the two disorders. To validate and further explore the result, expression levels of CREB3L1 in PRP were quantified by reverse-transcription quantitative PCR and compared among patients with ET, other Ph-MPNs, chronic myeloid leukemia (CML), and reactive hypercytosis and myelofibrosis. A CREB3L1 expression cutoff value determined based on PRP of 18 healthy volunteers accurately discriminated 150 driver mutation-positive Ph-MPNs from other entities (71 reactive hypercytosis and myelofibrosis, 6 CML, and 18 healthy volunteers) and showed both sensitivity and specificity of 1.0000. Importantly, CREB3L1 expression levels were significantly higher in ET compared with reactive thrombocytosis (P < .0001), and polycythemia vera compared with reactive erythrocytosis (P < .0001). Pathology-affirmed triple-negative ET (TN-ET) patients were divided into a high- and low-CREB3L1-expression group, and some patients in the low-expression group achieved a spontaneous remission during the clinical course. In conclusion, CREB3L1 analysis has the potential to single-handedly discriminate driver mutation-positive Ph-MPNs from reactive hypercytosis and myelofibrosis, and also may identify a subgroup within TN-ET showing distinct clinical features including spontaneous remission.

Scanning single-molecule counting system for Eprobe with highly simple and effective approach.

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

Deep phenotyping of myalgic encephalomyelitis/chronic fatigue syndrome in Japanese population.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex and debilitating disease with no molecular diagnostics and no treatment options. To identify potential markers of this illness, we profiled 48 patients and 52 controls for standard laboratory tests, plasma metabolomics, blood immuno-phenotyping and transcriptomics, and fecal microbiome analysis. Here, we identified a set of 26 potential molecular markers that distinguished ME/CFS patients from healthy controls. Monocyte number, microbiome abundance, and lipoprotein profiles appeared to be the most informative markers. When we correlated these molecular changes to sleep and cognitive measurements of fatigue, we found that lipoprotein and microbiome profiles most closely correlated with sleep disruption while a different set of markers correlated with a cognitive parameter. Sleep, lipoprotein, and microbiome changes occur early during the course of illness suggesting that these markers can be examined in a larger cohort for potential biomarker application. Our study points to a cluster of sleep-related molecular changes as a prominent feature of ME/CFS in our Japanese cohort.

Development of a Tet-On Inducible Expression System for the Anhydrobiotic Cell Line, Pv11.

The Pv11 cell line established from an African chironomid, Polypedilum vanderplanki, is the only cell line tolerant to complete desiccation. In Pv11 cells, a constitutive expression system for Pv11 cells was previously exploited and several reporter genes were successfully expressed. Here we report the identification of an effective minimal promoter for Pv11 cells and its application to the Tet-On inducible expression system. First, using a luciferase reporter assay, we showed that a 202 bp deletion fragment derived from the constitutively active 121-promoter functions in Pv11 cells as an appropriate minimal promoter with the Tet-On inducible expression system. The AcGFP1 (Aequorea coerulescens green fluorescent protein) was also successfully expressed in Pv11 cells using the inducible system. In addition to these reporter genes, the avian myeloblastosis virus reverse transcriptase α subunit (AMV RTα), which is one of the most widely commercially available RNA-dependent DNA polymerases, was successfully expressed through the inducible expression system and its catalytic activity was verified. These results demonstrate the establishment of an inducible expression system in cells that can be preserved in the dry state and highlight a possible application to the production of large and complex proteins.

Comparative transcriptomics of primary cells in vertebrates.

Gene expression profiles in homologous tissues have been observed to be different between species, which may be due to differences between species in the gene expression program in each cell type, but may also reflect differences in cell type composition of each tissue in different species. Here, we compare expression profiles in matching primary cells in human, mouse, rat, dog, and chicken using Cap Analysis Gene Expression (CAGE) and short RNA (sRNA) sequencing data from FANTOM5. While we find that expression profiles of orthologous genes in different species are highly correlated across cell types, in each cell type many genes were differentially expressed between species. Expression of genes with products involved in transcription, RNA processing, and transcriptional regulation was more likely to be conserved, while expression of genes encoding proteins involved in intercellular communication was more likely to have diverged during evolution. Conservation of expression correlated positively with the evolutionary age of genes, suggesting that divergence in expression levels of genes critical for cell function was restricted during evolution. Motif activity analysis showed that both promoters and enhancers are activated by the same transcription factors in different species. An analysis of expression levels of mature miRNAs and of primary miRNAs identified by CAGE revealed that evolutionary old miRNAs are more likely to have conserved expression patterns than young miRNAs. We conclude that key aspects of the regulatory network are conserved, while differential expression of genes involved in cell-to-cell communication may contribute greatly to phenotypic differences between species.

Comprehensive Characterization of Transcriptional Activity during Influenza A Virus Infection Reveals Biases in Cap-Snatching of Host RNA Sequences.

Macrophages in the lung detect and respond to influenza A virus (IAV), determining the nature of the immune response. Using terminal-depth cap analysis of gene expression (CAGE), we quantified transcriptional activity of both host and pathogen over a 24-h time course of IAV infection in primary human monocyte-derived macrophages (MDMs). This method allowed us to observe heterogenous host sequences incorporated into IAV mRNA, "snatched" 5' RNA caps, and corresponding RNA sequences from host RNAs. In order to determine whether cap-snatching is random or exhibits a bias, we systematically compared host sequences incorporated into viral mRNA ("snatched") against a complete survey of all background host RNA in the same cells, at the same time. Using a computational strategy designed to eliminate sources of bias due to read length, sequencing depth, and multimapping, we were able to quantify overrepresentation of host RNA features among the sequences that were snatched by IAV. We demonstrate biased snatching of numerous host RNAs, particularly small nuclear RNAs (snRNAs), and avoidance of host transcripts encoding host ribosomal proteins, which are required by IAV for replication. We then used a systems approach to describe the transcriptional landscape of the host response to IAV, observing many new features, including a failure of IAV-treated MDMs to induce feedback inhibitors of inflammation, seen in response to other treatments.IMPORTANCE Infection with influenza A virus (IAV) infection is responsible for an estimated 500,000 deaths and up to 5 million cases of severe respiratory illness each year. In this study, we looked at human primary immune cells (macrophages) infected with IAV. Our method allows us to look at both the host and the virus in parallel. We used these data to explore a process known as "cap-snatching," where IAV snatches a short nucleotide sequence from capped host RNA. This process was believed to be random. We demonstrate biased snatching of numerous host RNAs, including those associated with snRNA transcription, and avoidance of host transcripts encoding host ribosomal proteins, which are required by IAV for replication. We then describe the transcriptional landscape of the host response to IAV, observing new features, including a failure of IAV-treated MDMs to induce feedback inhibitors of inflammation, seen in response to other treatments.

CDK1 dependent phosphorylation of hTERT contributes to cancer progression.

The telomerase reverse transcriptase is upregulated in the majority of human cancers and contributes directly to cell transformation. Here we report that hTERT is phosphorylated at threonine 249 during mitosis by the serine/threonine kinase CDK1. Clinicopathological analyses reveal that phosphorylation of hTERT at threonine 249 occurs more frequently in aggressive cancers. Using CRISPR/Cas9 genome editing, we introduce substitution mutations at threonine 249 in the endogenous hTERT locus and find that phosphorylation of threonine 249 is necessary for hTERT-mediated RNA dependent RNA polymerase (RdRP) activity but dispensable for reverse transcriptase and terminal transferase activities. Cap Analysis of Gene Expression (CAGE) demonstrates that hTERT phosphorylation at 249 regulates the expression of specific genes that are necessary for cancer cell proliferation and tumor formation. These observations indicate that phosphorylation at threonine 249 regulates hTERT RdRP and contributes to cancer progression in a telomere independent manner.

MicroRNA-27a/b-3p and PPARG regulate SCAMP3 through a feed-forward loop during adipogenesis.

MicroRNAs (miRNA) modulate gene expression through feed-back and forward loops. Previous studies identified miRNAs that regulate transcription factors, including Peroxisome Proliferator Activated Receptor Gamma (PPARG), in adipocytes, but whether they influence adipogenesis via such regulatory loops remain elusive. Here we predicted and validated a novel feed-forward loop regulating adipogenesis and involved miR-27a/b-3p, PPARG and Secretory Carrier Membrane Protein 3 (SCAMP3). In this loop, expression of both PPARG and SCAMP3 was independently suppressed by miR-27a/b-3p overexpression. Knockdown of PPARG downregulated SCAMP3 expression at the late phase of adipogenesis, whereas reduction of SCAMP3 mRNA levels increased PPARG expression at early phase in differentiation. The latter was accompanied with upregulation of adipocyte-enriched genes, including ADIPOQ and FABP4, suggesting an anti-adipogenic role for SCAMP3. PPARG and SCAMP3 exhibited opposite behaviors regarding correlations with clinical phenotypes, including body mass index, body fat mass, adipocyte size, lipolytic and lipogenic capacity, and secretion of pro-inflammatory cytokines. While adipose PPARG expression was associated with more favorable metabolic phenotypes, SCAMP3 expression was linked to increased fat mass and insulin resistance. Together, we identified a feed-forward loop through which miR-27a/b-3p, PPARG and SCAMP3 cooperatively fine tune the regulation of adipogenesis, which potentially may impact whole body metabolism.