Molecular traces of Drosophila hemocytes reveal transcriptomic conservation with vertebrate myeloid cells.

Drosophila hemocytes serve as the primary defense system against harmful threats, allowing the animals to thrive. Hemocytes are often compared to vertebrate innate immune system cells due to the observed functional similarities between the two. However, the similarities have primarily been established based on a limited number of genes and their functional homologies. Thus, a systematic analysis using transcriptomic data could offer novel insights into Drosophila hemocyte function and provide new perspectives on the evolution of the immune system. Here, we performed cross-species comparative analyses using single-cell RNA sequencing data from Drosophila and vertebrate immune cells. We found several conserved markers for the cluster of differentiation (CD) genes in Drosophila hemocytes and validated the role of CG8501 (CD59) in phagocytosis by plasmatocytes, which function much like macrophages in vertebrates. By comparing whole transcriptome profiles in both supervised and unsupervised analyses, we showed that Drosophila hemocytes are largely homologous to vertebrate myeloid cells, especially plasmatocytes to monocytes/macrophages and prohemocyte 1 (PH1) to hematopoietic stem cells. Furthermore, a small subset of prohemocytes with hematopoietic potential displayed homology with hematopoietic progenitor populations in vertebrates. Overall, our results provide a deeper understanding of molecular conservation in the Drosophila immune system.

Clustering malignant cell states using universally variable genes.

Single-cell RNA sequencing (scRNA-seq) has revealed important insights into the heterogeneity of malignant cells. However, sample-specific genomic alterations often confound such analysis, resulting in patient-specific clusters that are difficult to interpret. Here, we present a novel approach to address the issue. By normalizing gene expression variances to identify universally variable genes (UVGs), we were able to reduce the formation of sample-specific clusters and identify underlying molecular hallmarks in malignant cells. In contrast to highly variable genes vulnerable to a specific sample bias, UVGs led to better detection of clusters corresponding to distinct malignant cell states. Our results demonstrate the utility of this approach for analyzing scRNA-seq data and suggest avenues for further exploration of malignant cell heterogeneity.

Toward the functional interpretation of somatic structural variations: bulk- and single-cell approaches.

Structural variants (SVs) are genomic rearrangements that can take many different forms such as copy number alterations, inversions and translocations. During cell development and aging, somatic SVs accumulate in the genome with potentially neutral, deleterious or pathological effects. Generation of somatic SVs is a key mutational process in cancer development and progression. Despite their importance, the detection of somatic SVs is challenging, making them less studied than somatic single-nucleotide variants. In this review, we summarize recent advances in whole-genome sequencing (WGS)-based approaches for detecting somatic SVs at the tissue and single-cell levels and discuss their advantages and limitations. First, we describe the state-of-the-art computational algorithms for somatic SV calling using bulk WGS data and compare the performance of somatic SV detectors in the presence or absence of a matched-normal control. We then discuss the unique features of cutting-edge single-cell-based techniques for analyzing somatic SVs. The advantages and disadvantages of bulk and single-cell approaches are highlighted, along with a discussion of their sensitivity to copy-neutral SVs, usefulness for functional inferences and experimental and computational costs. Finally, computational approaches for linking somatic SVs to their functional readouts, such as those obtained from single-cell transcriptome and epigenome analyses, are illustrated, with a discussion of the promise of these approaches in health and diseases.

Tumor immune microenvironment lncRNAs.

Long non-coding ribonucleic acids (RNAs) (lncRNAs) are key players in tumorigenesis and immune responses. The nature of their cell type-specific gene expression and other functional evidence support the idea that lncRNAs have distinct cellular functions in the tumor immune microenvironment (TIME). To date, the majority of lncRNA studies have heavily relied on bulk RNA-sequencing data in which various cell types contribute to an averaged signal, limiting the discovery of cell type-specific lncRNA functions. Single-cell RNA-sequencing (scRNA-seq) is a potential solution for tackling this limitation despite the lack of annotations for low abundance yet cell type-specific lncRNAs. Hence, updated annotations and further understanding of the cellular expression of lncRNAs will be necessary for characterizing cell type-specific functions of lncRNA genes in the TIME. In this review, we discuss lncRNAs that are specifically expressed in tumor and immune cells, summarize the regulatory functions of the lncRNAs at the cell type level and highlight how a scRNA-seq approach can help to study the cell type-specific functions of TIME lncRNAs.

Comparison of Proportional Mortality Between Korean Atomic Bomb Survivors and the General Population During 1992-2019.

BACKGROUND: Atomic bombs dropped on Hiroshima and Nagasaki in Japan in August 1945 were estimated to have killed approximately 70,000 Koreans. In Japan, studies on the health status and mortality of atomic bomb survivors compared with the non-exposed population have been conducted. However, there have been no studies related to the mortality of Korean atomic bomb survivors. Therefore, we aimed to study the cause of death of atomic bomb survivors compared to that of the general population. METHODS: Of 2,299 atomic bomb survivors registered with the Korean Red Cross, 2,176 were included in the study. In the general population, the number of deaths by age group was calculated from 1992 to 2019, and 6,377,781 individuals were assessed. Causes of death were categorized according to the Korean Standard Classification of Diseases. To compare the proportional mortality between the two groups, the P value for the ratio test was confirmed, and the Cochran-Armitage trend test and χ² test were performed to determine the cause of death according to the distance from the hypocenter. RESULTS: Diseases of the circulatory system were the most common cause of death (25.4%), followed by neoplasms (25.1%) and diseases of the respiratory system (10.6%) in atomic bomb survivors who died between 1992 and 2019. The proportional mortality associated with respiratory diseases, nervous system diseases, and other diseases among atomic bomb survivors was higher than that of the general population. Of the dead people between 1992 and 2019, the age at death of survivors who were exposed at a close distance was younger than those who were exposed at a greater distance. CONCLUSION: Overall, proportional mortality of respiratory diseases and nervous system diseases was high in atomic bomb survivors, compared with the general population. Further studies on the health status of Korean atomic bomb survivors are needed.

Global analyses of the effect of different cellular contexts on microRNA targeting.

MicroRNA (miRNA) regulation clearly impacts animal development, but the extent to which development-with its resulting diversity of cellular contexts-impacts miRNA regulation is unclear. Here, we compared cohorts of genes repressed by the same miRNAs in different cell lines and tissues and found that target repertoires were largely unaffected, with secondary effects explaining most of the differential responses detected. Outliers resulting from differential direct targeting were often attributable to alternative 3' UTR isoform usage that modulated the presence of miRNA sites. More inclusive examination of alternative 3' UTR isoforms revealed that they influence ∼10% of predicted targets when comparing any two cell types. Indeed, considering alternative 3' UTR isoform usage improved prediction of targeting efficacy significantly beyond the improvements observed when considering constitutive isoform usage. Thus, although miRNA targeting is remarkably consistent in different cell types, considering the 3' UTR landscape helps predict targeting efficacy and explain differential regulation that is observed.

HERES, a lncRNA that regulates canonical and noncanonical Wnt signaling pathways via interaction with EZH2.

Wnt signaling through both canonical and noncanonical pathways plays a core role in development. Dysregulation of these pathways often causes cancer development and progression. Although the pathways independently contribute to the core processes, a regulatory molecule that commonly activates both of them has not yet been reported. Here, we describe a long noncoding RNA (lncRNA), HERES, that epigenetically regulates both canonical and noncanonical Wnt signaling pathways in esophageal squamous cell carcinoma (ESCC). For this study, we performed RNA-seq analysis on Korean ESCC patients and validated these results on a larger ESCC cohort to identify lncRNAs commonly dysregulated in ESCCs. Six of the dysregulated lncRNAs were significantly associated with the clinical outcomes of ESCC patients and defined 4 ESCC subclasses with different prognoses. HERES reduction repressed cell proliferation, migration, invasion, and colony formation in ESCC cell lines and tumor growth in xenograft models. HERES appears to be a transacting factor that regulates CACNA2D3, SFRP2, and CXXC4 simultaneously to activate Wnt signaling pathways through an interaction with EZH2 via its G-quadruple structure-like motif. Our results suggest that HERES holds substantial potential as a therapeutic target for ESCC and probably other cancers caused by defects in Wnt signaling pathways.

The small peptide world in long noncoding RNAs.

Long noncoding RNAs (lncRNAs) are a group of transcripts that are longer than 200 nucleotides (nt) without coding potential. Over the past decade, tens of thousands of novel lncRNAs have been annotated in animal and plant genomes because of advanced high-throughput RNA sequencing technologies and with the aid of coding transcript classifiers. Further, a considerable number of reports have revealed the existence of stable, functional small peptides (also known as micropeptides), translated from lncRNAs. In this review, we discuss the methods of lncRNA classification, the investigations regarding their coding potential and the functional significance of the peptides they encode.

Assessment of Inflammation in Pulmonary Artery Hypertension by 68Ga-Mannosylated Human Serum Albumin.

Rationale: Diagnosis and monitoring of patients with pulmonary artery hypertension (PAH) is currently difficult.Objectives: We aimed to develop a noninvasive imaging modality for PAH that tracks the infiltration of macrophages into the pulmonary vasculature, using a positron emission tomography (PET) agent, 68Ga-2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) mannosylated human serum albumin (MSA), that targets the mannose receptor (MR).Methods: We induced PAH in rats by monocrotaline injection. Tissue analysis, echocardiography, and 68Ga-NOTA-MSA PET were performed weekly in rats after monocrotaline injection and in those treated with either sildenafil or macitentan. The translational potential of 68Ga-NOTA-MSA PET was explored in patients with PAH.Measurements and Main Results: Gene sets related to macrophages were significantly enriched on whole transcriptome sequencing of the lung tissue in PAH rats. Serial PET images of PAH rats demonstrated increasing uptake of 68Ga-NOTA-MSA in the lung by time that corresponded with the MR-positive macrophage recruitment observed in immunohistochemistry. In sildenafil- or macitentan-treated PAH rats, the infiltration of MR-positive macrophages by histology and the uptake of 68Ga-NOTA-MSA on PET was significantly lower than that of the PAH-only group. The pulmonary uptake of 68Ga-NOTA-MSA was significantly higher in patients with PAH than normal subjects (P = 0.009) or than those with pulmonary hypertension by left heart disease (P = 0.019) (n = 5 per group).Conclusions:68Ga-NOTA-MSA PET can help diagnose PAH and monitor the inflammatory status by imaging the degree of macrophage infiltration into the lung. These observations suggest that 68Ga-NOTA-MSA PET has the potential to be used as a novel noninvasive diagnostic and monitoring tool of PAH.

En bloc and segmental deletions of human XIST reveal X chromosome inactivation-involving RNA elements.

The XIST RNA is a non-coding RNA that induces X chromosome inactivation (XCI). Unlike the mouse Xist RNA, how the human XIST RNA controls XCI in female cells is less well characterized, and its functional motifs remain unclear. To systematically decipher the XCI-involving elements of XIST RNA, 11 smaller XIST segments, including repeats A, D and E; human-specific repeat elements; the promoter; and non-repetitive exons, as well as the entire XIST gene, were homozygously deleted in K562 cells using the Cas9 nuclease and paired guide RNAs at high efficiencies, followed by high-throughput RNA sequencing and RNA fluorescence in situ hybridization experiments. Clones containing en bloc and promoter deletions that consistently displayed no XIST RNAs and a global up-regulation of X-linked genes confirmed that the deletion of XIST reactivates the inactive X chromosome. Systematic analyses of segmental deletions delineated that exon 5 harboring the non-repeat element is important for X-inactivation maintenance, whereas exons 2, 3 and 4 as well as the other repeats in exon 1 are less important, a different situation from that of mouse Xist. This Cas9-assisted dissection of XIST allowed us to understand the unique functional domains within the human XIST RNA.

High-confidence coding and noncoding transcriptome maps.

The advent of high-throughput RNA sequencing (RNA-seq) has led to the discovery of unprecedentedly immense transcriptomes encoded by eukaryotic genomes. However, the transcriptome maps are still incomplete partly because they were mostly reconstructed based on RNA-seq reads that lack their orientations (known as unstranded reads) and certain boundary information. Methods to expand the usability of unstranded RNA-seq data by predetermining the orientation of the reads and precisely determining the boundaries of assembled transcripts could significantly benefit the quality of the resulting transcriptome maps. Here, we present a high-performing transcriptome assembly pipeline, called CAFE, that significantly improves the original assemblies, respectively assembled with stranded and/or unstranded RNA-seq data, by orienting unstranded reads using the maximum likelihood estimation and by integrating information about transcription start sites and cleavage and polyadenylation sites. Applying large-scale transcriptomic data comprising 230 billion RNA-seq reads from the ENCODE, Human BodyMap 2.0, The Cancer Genome Atlas, and GTEx projects, CAFE enabled us to predict the directions of about 220 billion unstranded reads, which led to the construction of more accurate transcriptome maps, comparable to the manually curated map, and a comprehensive lncRNA catalog that includes thousands of novel lncRNAs. Our pipeline should not only help to build comprehensive, precise transcriptome maps from complex genomes but also to expand the universe of noncoding genomes.

Mass Cytometry and Single-Cell RNA-seq Profiling of the Heterogeneity in Human Peripheral Blood Mononuclear Cells Interacting with Silver Nanoparticles.

Understanding the interactions between nanoparticles (NPs) and human immune cells is necessary for justifying their utilization in consumer products and biomedical applications. However, conventional assays may be insufficient in describing the complexity and heterogeneity of cell-NP interactions. Herein, mass cytometry and single-cell RNA-sequencing (scRNA-seq) are complementarily used to investigate the heterogeneous interactions between silver nanoparticles (AgNPs) and primary immune cells. Mass cytometry reveals the heterogeneous biodistribution of the positively charged polyethylenimine-coated AgNPs in various cell types and finds that monocytes and B cells have higher association with the AgNPs than other populations. scRNA-seq data of these two cell types demonstrate that each type has distinct responses to AgNP treatment: NRF2-mediated oxidative stress is confined to B cells, whereas monocytes show Fcγ-mediated phagocytosis. Besides the between-population heterogeneity, analysis of single-cell dose-response relationships further reveals within-population diversity for the B cells and naïve CD4+ T cells. Distinct subsets having different levels of cellular responses with respect to their cellular AgNP doses are found. This study demonstrates that the complementary use of mass cytometry and scRNA-seq is helpful for gaining in-depth knowledge on the heterogeneous interactions between immune cells and NPs and can be incorporated into future toxicity assessments of nanomaterials.

In vivo high-throughput profiling of CRISPR-Cpf1 activity.

CRISPR from Prevotella and Francisella 1 (Cpf1) is an effector endonuclease of the class 2 CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) gene editing system. We developed a method for evaluating Cpf1 activity, based on target sequence composition in mammalian cells, in a high-throughput manner. A library of >11,000 target sequence and guide RNA pairs was delivered into human cells using lentiviral vectors. Subsequent delivery of Cpf1 into this cell library induced insertions and deletions (indels) at the integrated synthetic target sequences, which allowed en masse evaluation of Cpf1 activity by using deep sequencing. With this approach, we determined protospacer-adjacent motif sequences of two Cpf1 nucleases, one from Acidaminococcus sp. BV3L6 (hereafter referred to as AsCpf1) and the other from Lachnospiraceae bacterium ND2006 (hereafter referred to as LbCpf1). We also defined target-sequence-dependent activity profiles of AsCpf1, which enabled the development of a web tool that predicts the indel frequencies for given target sequences (http://big.hanyang.ac.kr/cindel). Both the Cpf1 characterization profile and the in vivo high-throughput evaluation method will greatly facilitate Cpf1-based genome editing.

The present and future of de novo whole-genome assembly.

As the advent of next-generation sequencing (NGS) technology, various de novo assembly algorithms based on the de Bruijn graph have been developed to construct chromosome-level sequences. However, numerous technical or computational challenges in de novo assembly still remain, although many bright ideas and heuristics have been suggested to tackle the challenges in both experimental and computational settings. In this review, we categorize de novo assemblers on the basis of the type of de Bruijn graphs (Hamiltonian and Eulerian) and discuss the challenges of de novo assembly for short NGS reads regarding computational complexity and assembly ambiguity. Then, we discuss how the limitations of the short reads can be overcome by using a single-molecule sequencing platform that generates long reads of up to several kilobases. In fact, the long read assembly has caused a paradigm shift in whole-genome assembly in terms of algorithms and supporting steps. We also summarize (i) hybrid assemblies using both short and long reads and (ii) overlap-based assemblies for long reads and discuss their challenges and future prospects. This review provides guidelines to determine the optimal approach for a given input data type, computational budget or genome.

HuR stabilizes a polyadenylated form of replication-dependent histone mRNAs under stress conditions.

All metazoan mRNAs have a poly(A) tail at the 3' end with the exception of replication-dependent histone (RDH) mRNAs, which end in a highly conserved stem-loop (SL) structure. However, a subset of RDH mRNAs are reported to be polyadenylated under physiologic conditions. The molecular details of the biogenesis of polyadenylated RDH [poly(A)+ RDH] mRNAs remain unknown. In this study, our genome-wide analyses reveal that puromycin treatment or UVC irradiation stabilizes poly(A)+ RDH mRNAs, relative to canonical RDH mRNAs, which end in an SL structure. We demonstrate that the stabilization of poly(A)+ RDH mRNAs occurs in a translation-independent manner and is regulated via human antigen R (HuR) binding to the extended 3' UTR under stress conditions. Our data suggest that HuR regulates the expression of poly(A)+ RDH mRNAs.-Ryu, I., Park, Y., Seo, J.-W., Park, O. H., Ha, H., Nam, J.-W., Kim, Y. K. HuR stabilizes a polyadenylated form of replication-dependent histone mRNAs under stress conditions.

Transcriptome Analysis Reveals Nonfoamy Rather Than Foamy Plaque Macrophages Are Proinflammatory in Atherosclerotic Murine Models.

RATIONALE: Monocyte infiltration into the subintimal space and its intracellular lipid accumulation are the most prominent features of atherosclerosis. To understand the pathophysiology of atherosclerotic disease, we need to understand the characteristics of lipid-laden foamy macrophages in the subintimal space during atherosclerosis. OBJECTIVE: We sought to examine the transcriptomic profiles of foamy and nonfoamy macrophages isolated from atherosclerotic intima. METHODS AND RESULTS: Single-cell RNA sequencing analysis of CD45+ leukocytes from murine atherosclerotic aorta revealed that there are macrophage subpopulations with distinct differentially expressed genes involved in various functional pathways. To specifically characterize the intimal foamy macrophages of plaque, we developed a lipid staining-based flow cytometric method for analyzing the lipid-laden foam cells of atherosclerotic aortas. We used the fluorescent lipid probe BODIPY493/503 and assessed side-scattered light as an indication of cellular granularity. BODIPYhiSSChi foamy macrophages were found residing in intima and expressing CD11c. Foamy macrophage accumulation determined by flow cytometry was positively correlated with the severity of atherosclerosis. Bulk RNA sequencing analysis showed that compared with nonfoamy macrophages, foamy macrophages expressed few inflammatory genes but many lipid-processing genes. Intimal nonfoamy macrophages formed the major population expressing IL (interleukin)-1ß and many other inflammatory transcripts in atherosclerotic aorta. CONCLUSIONS: RNA sequencing analysis of intimal macrophages from atherosclerotic aorta revealed that lipid-loaded plaque macrophages are not likely the plaque macrophages that drive lesional inflammation.

DROSHA targets its own transcript to modulate alternative splicing.

The nuclear RNase III enzyme DROSHA interacts with its cofactor DGCR8 to form the Microprocessor complex, which initiates microRNA (miRNA) maturation by cleaving hairpin structures embedded in primary transcripts. Apart from its central role in the biogenesis of miRNAs, DROSHA is also known to recognize and cleave miRNA-like hairpins in a subset of transcripts without apparent small RNA production. Here, we report that the human DROSHA transcript is one such noncanonical target of DROSHA. Mammalian DROSHA genes have evolved a conserved hairpin structure spanning a specific exon-intron junction, which serves as a substrate for the Microprocessor in human cells but not in murine cells. We show that it is this hairpin element that decides whether the overlapping exon is alternatively or constitutively spliced. We further demonstrate that DROSHA promotes skipping of the overlapping exon in human cells independently of its cleavage function. Our findings add to the expanding list of noncanonical DROSHA functions.

TERIUS: accurate prediction of lncRNA via high-throughput sequencing data representing RNA-binding protein association.

BACKGROUND: LncRNAs are long regulatory non-coding RNAs, some of which are arguably predicted to have coding potential. Despite coding potential classifiers that utilize ribosome profiling data successfully detected actively translated regions, they are less sensitive to lncRNAs. Furthermore, lncRNA annotation can be susceptible to false positives obtained from 3' untranslated region (UTR) fragments of mRNAs. RESULTS: To lower these limitations in lncRNA annotation, we present a novel tool TERIUS that provides a two-step filtration process to distinguish between bona fide and false lncRNAs. The first step successfully separates lncRNAs from protein-coding genes showing enhanced sensitivity compared to other methods. To eliminate 3'UTR fragments, the second step takes advantage of the 3'UTR-specific association with regulator of nonsense transcripts 1 (UPF1), leading to refined lncRNA annotation. Importantly, TERIUS enabled the detection of misclassified transcripts in published lncRNA annotations. CONCLUSIONS: TERIUS is a robust method for lncRNA annotation, which provides an additional filtration step for 3'UTR fragments. TERIUS was able to successfully re-classify GENCODE and miTranscriptome lncRNA annotations. We believe that TERIUS can benefit construction of extensive and accurate non-coding transcriptome maps in many genomes.

Expanding the microRNA targeting code: functional sites with centered pairing.

Most metazoan microRNA (miRNA) target sites have perfect pairing to the seed region, located near the miRNA 5' end. Although pairing to the 3' region sometimes supplements seed matches or compensates for mismatches, pairing to the central region has been known to function only at rare sites that impart Argonaute-catalyzed mRNA cleavage. Here, we present "centered sites," a class of miRNA target sites that lack both perfect seed pairing and 3'-compensatory pairing and instead have 11-12 contiguous Watson-Crick pairs to the center of the miRNA. Although centered sites can impart mRNA cleavage in vitro (in elevated Mg(2+)), in cells they repress protein output without consequential Argonaute-catalyzed cleavage. Our study also identified extensively paired sites that are cleavage substrates in cultured cells and human brain. This expanded repertoire of cleavage targets and the identification of the centered site type help explain why central regions of many miRNAs are evolutionarily conserved.

Non-Coding Transcriptome Maps across Twenty Tissues of the Korean Black Chicken, Yeonsan Ogye.

Yeonsan Ogye is a rare Korean domestic chicken breed whose entire body, including feathers and skin, has a unique black coloring. Although some protein-coding genes related to this unique feature have been examined, non-coding elements have not been widely investigated. Thus, we evaluated coding and non-coding transcriptome expression and identified long non-coding RNAs functionally linked to protein-coding genes in Ogye. High-throughput RNA sequencing and DNA methylation sequencing were performed to profile the expression of 14,264 Ogye protein-coding and 6900 long non-coding RNA (lncRNA) genes and detect DNA methylation in 20 different tissues of an individual Ogye. Approximately 75% of Ogye lncRNAs and 45% of protein-coding genes showed tissue-specific expression. For some genes, tissue-specific expression levels were inversely correlated with DNA methylation levels in their promoters. Approximately 39% of tissue-specific lncRNAs displayed functional associations with proximal or distal protein-coding genes. Heat shock transcription factor 2-associated lncRNAs appeared to be functionally linked to protein-coding genes specifically expressed in black skin tissues, more syntenically conserved in mammals, and differentially expressed in black relative to in white tissues. Pending experimental validation, our findings increase the understanding of how the non-coding genome regulates unique phenotypes and can be used for future genomic breeding of chickens.