circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA.

Circular RNAs (circRNAs) represent a class of widespread endogenous RNAs that regulate gene expression and thereby influence cell biological decisions with implications for the pathogenesis of several diseases. Here, we disclose a novel gene-regulatory role of circHIPK3 by combining analyses of large genomics datasets and mechanistic cell biological follow-up experiments. Specifically, we use temporal depletion of circHIPK3 or specific RNA binding proteins (RBPs) and identify several perturbed genes by RNA sequencing analyses. Using expression-coupled motif analyses of mRNA expression data from various knockdown experiments, we identify an 11-mer motif within circHIPK3, which is also enriched in genes that become downregulated upon circHIPK3 depletion. By mining eCLIP datasets, we find that the 11-mer motif constitutes a strong binding site for IGF2BP2 and validate this circHIPK3-IGF2BP2 interaction experimentally using RNA-immunoprecipitation and competition assays in bladder cancer cell lines. Our results suggest that circHIPK3 and IGF2BP2 mRNA targets compete for binding. Since the identified 11-mer motif found in circHIPK3 is enriched in upregulated genes following IGF2BP2 knockdown, and since IGF2BP2 depletion conversely globally antagonizes the effect of circHIPK3 knockdown on target genes, our results suggest that circHIPK3 can sequester IGF2BP2 as a competing endogenous RNA (ceRNA), leading to target mRNA stabilization. As an example of a circHIPK3-regulated gene, we focus on the STAT3 mRNA as a specific substrate of IGF2BP2 and validate that manipulation of circHIPK3 regulates IGF2BP2-STAT3 mRNA binding and thereby STAT3 mRNA levels. However, absolute copy number quantifications demonstrate that IGF2BP2 outnumbers circHIPK3 by orders of magnitude, which is inconsistent with a simple 1:1 ceRNA hypothesis. Instead, we show that circHIPK3 can nucleate multiple copies of IGF2BP2, potentially via phase separation, to produce IGF2BP2 condensates. Finally, we show that circHIPK3 expression correlates with overall survival of patients with bladder cancer. Our results are consistent with a model where relatively few cellular circHIPK3 molecules function as inducers of IGF2BP2 condensation thereby regulating STAT3 and other key factors for cell proliferation and potentially cancer progression.

Computational Methods for Single-Cell Proteomics.

Advances in single-cell proteomics technologies have resulted in high-dimensional datasets comprising millions of cells that are capable of answering key questions about biology and disease. The advent of these technologies has prompted the development of computational tools to process and visualize the complex data. In this review, we outline the steps of single-cell and spatial proteomics analysis pipelines. In addition to describing available methods, we highlight benchmarking studies that have identified advantages and pitfalls of the currently available computational toolkits. As these technologies continue to advance, robust analysis tools should be developed in tandem to take full advantage of the potential biological insights provided by these data.

Sex chromosome aneuploidies give rise to changes in the circular RNA profile: A circular transcriptome-wide study of Turner and Klinefelter syndrome across different tissues.

Purpose: The landscape of circular RNAs (circRNAs), an important class of non-coding RNAs that regulate gene expression, has never been described in human disorders of sex chromosome aneuploidies. We profiled circRNAs in Turner syndrome females (45,X; TS) and Klinefelter syndrome males (47,XXY; KS) to investigate how circRNAs respond to a missing or an extra X chromosome. Methods: Samples of blood, muscle and fat were collected from individuals with TS (n = 33) and KS (n = 22) and from male (n = 16) and female (n = 44) controls. CircRNAs were identified using a combination of circRNA identification pipelines (CIRI2, CIRCexplorer2 and circRNA_finder). Results: Differential expression of circRNAs was observed throughout the genome in TS and KS, in all tissues. The host-genes from which several of these circRNAs were derived, were associated with known phenotypic traits. Furthermore, several differentially expressed circRNAs had the potential to capture micro RNAs that targeted protein-coding genes with altered expression in TS and KS. Conclusion: Sex chromosome aneuploidies introduce changes in the circRNA transcriptome, demonstrating that the genomic changes in these syndromes are more complex than hitherto thought. CircRNAs may help explain some of the genomic and phenotypic traits observed in these syndromes.

Mass cytometry reveals a conserved immune trajectory of recovery in hospitalized COVID-19 patients.

While studies have elucidated many pathophysiological elements of COVID-19, little is known about immunological changes during COVID-19 resolution. We analyzed immune cells and phosphorylated signaling states at single-cell resolution from longitudinal blood samples of patients hospitalized with COVID-19, pneumonia and/or sepsis, and healthy individuals by mass cytometry. COVID-19 patients showed distinct immune compositions and an early, coordinated, and elevated immune cell signaling profile associated with early hospital discharge. Intra-patient longitudinal analysis revealed changes in myeloid and T cell frequencies and a reduction in immune cell signaling across cell types that accompanied disease resolution and discharge. These changes, together with increases in regulatory T cells and reduced signaling in basophils, also accompanied recovery from respiratory failure and were associated with better outcomes at time of admission. Therefore, although patients have heterogeneous immunological baselines and highly variable disease courses, a core immunological trajectory exists that defines recovery from severe SARS-CoV-2 infection.

A conserved immune trajectory of recovery in hospitalized COVID-19 patients.

Many studies have provided insights into the immune response to COVID-19; however, little is known about the immunological changes and immune signaling occurring during COVID-19 resolution. Individual heterogeneity and variable disease resolution timelines obscure unifying immune characteristics. Here, we collected and profiled >200 longitudinal peripheral blood samples from patients hospitalized with COVID-19, with other respiratory infections, and healthy individuals, using mass cytometry to measure immune cells and signaling states at single cell resolution. COVID-19 patients showed a unique immune composition and an early, coordinated and elevated immune cell signaling profile, which correlated with early hospital discharge. Intra-patient time course analysis tied to clinically relevant events of recovery revealed a conserved set of immunological processes that accompany, and are unique to, disease resolution and discharge. This immunological process, together with additional changes in CD4 regulatory T cells and basophils, accompanies recovery from respiratory failure and is associated with better clinical outcomes at the time of admission. Our work elucidates the biological timeline of immune recovery from COVID-19 and provides insights into the fundamental processes of COVID-19 resolution in hospitalized patients.

The transcriptional landscape and biomarker potential of circular RNAs in prostate cancer.

BACKGROUND: Circular RNAs (circRNAs) constitute a largely unexplored source for biomarker discovery in prostate cancer (PC). Here, we characterize the biomarker potential of circRNAs in PC, where the need for novel diagnostic and prognostic tools to facilitate more personalized management is pressing. METHODS: We profiled the transcriptomic landscape of circRNAs in PC by total RNA sequencing of 31 adjacent-normal and 143 tumor samples from localized (radical prostatectomy (RP)) and metastatic PC patients (cohort 1, training). Diagnostic and prognostic potential was evaluated in cohort 1, and 39 top circRNA candidates were selected for validation in two additional PC cohorts (cohort 2, n = 111; RP cohort 3, n = 191) by NanoString-based expression analysis. Biochemical recurrence (BCR)-free survival was assessed using Kaplan-Meier, univariate, and multivariate Cox regression analyses. The circRNA candidates were further detected in extracellular vesicle (EV)-enriched plasma samples from PC patients and controls (cohort 4, n = 54). RESULTS: Expression of circABCC4, circFAT3, circATRNL1, and circITGA7 was highly cancer-specific (area under the curve 0.71-0.86), while low circITGA7 expression was significantly (P < 0.05) associated with BCR in univariate analysis in two RP cohorts. Moreover, we successfully trained and validated a novel 5-circRNA prognostic signature (circKMD1A/circTULP4/circZNF532/circSUMF1/circMKLN1) significantly associated with BCR beyond routine clinicopathological variables (RP cohort 1: P = 0.02, hazard ratio = 2.1; RP cohort 3: P < 0.001, hazard ratio = 2.1). Lastly, we provide proof-of-principle for detection of candidate circRNAs in EV-enriched plasma samples from PC patients. CONCLUSIONS: circRNAs hold great biomarker potential in PC and display both high cancer specificity and association to disease progression.

Transcriptome-wide profiles of circular RNA and RNA-binding protein interactions reveal effects on circular RNA biogenesis and cancer pathway expression.

BACKGROUND: Circular RNAs (circRNAs) are stable, often highly expressed RNA transcripts with potential to modulate other regulatory RNAs. A few circRNAs have been shown to bind RNA-binding proteins (RBPs); however, little is known about the prevalence and distribution of these interactions in different biological contexts. METHODS: We conduct an extensive screen of circRNA-RBP interactions in the ENCODE cell lines HepG2 and K562. We profile circRNAs in deep-sequenced total RNA samples and analyze circRNA-RBP interactions using a large set of eCLIP data with binding sites of 150 RBPs. We validate interactions for select circRNAs and RBPs by performing RNA immunoprecipitation and functionally characterize our most interesting candidates by conducting knockdown studies followed by RNA-Seq. RESULTS: We generate a comprehensive catalog of circRNA-RBP interactions in HepG2 and K562 cells. We show that KHSRP binding sites are enriched in flanking introns of circRNAs and that KHSRP depletion affects circRNA biogenesis. We identify circRNAs that are highly covered by RBP binding sites and experimentally validate individual circRNA-RBP interactions. We show that circCDYL, a highly expressed circRNA with clinical and functional implications in bladder cancer, is almost completely covered with GRWD1 binding sites in HepG2 cells, and that circCDYL depletion counteracts the effect of GRWD1 depletion. Furthermore, we confirm interactions between circCDYL and RBPs in bladder cancer cells and demonstrate that circCDYL depletion affects hallmarks of cancer and perturbs the expression of key cancer genes, e.g., TP53. Finally, we show that elevated levels of circCDYL are associated with overall survival of bladder cancer patients. CONCLUSIONS: Our study demonstrates transcriptome-wide and cell-type-specific circRNA-RBP interactions that could play important regulatory roles in tumorigenesis.

Molecular correlates of cisplatin-based chemotherapy response in muscle invasive bladder cancer by integrated multi-omics analysis.

Overtreatment with cisplatin-based chemotherapy is a major issue in the management of muscle-invasive bladder cancer (MIBC), and currently none of the reported biomarkers for predicting response have been implemented in the clinic. Here we perform a comprehensive multi-omics analysis (genomics, transcriptomics, epigenomics and proteomics) of 300 MIBC patients treated with chemotherapy (neoadjuvant or first-line) to identify molecular changes associated with treatment response. DNA-based associations with response converge on genomic instability driven by a high number of chromosomal alterations, indels, signature 5 mutations and/or BRCA2 mutations. Expression data identifies the basal/squamous gene expression subtype to be associated with poor response. Immune cell infiltration and high PD-1 protein expression are associated with treatment response. Through integration of genomic and transcriptomic data, we demonstrate patient stratification to groups of low and high likelihood of cisplatin-based response. This could pave the way for future patient selection following validation in prospective clinical trials.

High-throughput RNA sequencing from paired lesional- and non-lesional skin reveals major alterations in the psoriasis circRNAome.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes. It is one of the most prevalent chronic inflammatory skin conditions in adults worldwide, with a considerable negative impact on quality of life. Circular RNAs (circRNAs) are a recently identified type of non-coding RNA with diverse cellular functions related to their exceptional stability. In particular, some circRNAs can bind and regulate microRNAs (miRNAs), a group of RNAs that play a role in the pathogenesis of psoriasis. The aim of this study was to characterize the circRNAome in psoriasis and to assess potential correlations to miRNA expression patterns. METHODS: We used high-throughput RNA-sequencing (RNA-seq), NanoString nCounter technology and RNA chromogenic in situ hybridization (CISH) to profile the circRNA expression in paired lesional and non-lesional psoriatic skin from patients with psoriasis vulgaris. In addition, 799 miRNAs were profiled using NanoString nCounter technology and laser capture microdissection was used to study the dermis and epidermis separately. RESULTS: We found a substantial down-regulation of circRNA expression in lesional skin compared to non-lesional skin. We observed that this mainly applies to the epidermis by analyzing laser capture microdissected tissues. We also found that the majority of the circRNAs were downregulated independently of their corresponding linear host genes. The observed downregulation of circRNAs in psoriasis was neither due to altered expression levels of factors known to affect circRNA biogenesis, nor because lesional skin contained an increased number of inflammatory cells such as lymphocytes. Finally, we observed that the overall differences in available miRNA binding sites on the circRNAs between lesional and non-lesional skin did not correlate with differences in miRNA expression patterns. CONCLUSIONS: We have performed the first genome-wide circRNA profiling of paired lesional and non-lesional skin from patients with psoriasis and revealed that circRNAs are much less abundant in the lesional samples. Whether this is a cause or a consequence of the disease remains to be revealed, however, we found no evidence that the loss of miRNA binding sites on the circRNAs could explain differences in miRNA expression between lesional and non-lesional skin.

Circular RNAs are abundantly expressed and upregulated during human epidermal stem cell differentiation.

The expression patterns of endogenous circular RNA (circRNA) molecules during epidermal stem cell (EpSC) differentiation have not previously been explored. Here, we show that circRNAs are abundantly expressed in EpSCs and that their expression change dramatically during differentiation in a coordinated manner. Overall, circRNAs are expressed at higher levels in the differentiated cells, and many upregulated circRNAs are derived from developmental genes, including four different circRNAs from DLG1. The observed changes in circRNA expression were largely independent of host gene expression, and circRNAs independently upregulated upon differentiation are more prone to AGO2 binding and have more predicted miRNA binding sites compared to stably expressed circRNAs. In particular, upregulated circRNAs from the HECTD1 and ZNF91 genes have exceptionally high numbers of AGO2 binding sites and predicted miRNA target sites, and circZNF91 contains 24 target sites for miR-23b-3p, which is known to play important roles in keratinocyte differentiation. We also observed that upregulated circRNAs are less likely to be flanked by homologues inverted Alu repeats compared to stably expressed circRNAs. This coincide with DHX9 being upregulated in the differentiated keratinocytes. Finally, none of the circRNAs upregulated upon differentiation were also upregulated upon DNMT3A or DNMT3B knockdown, making it unlikely that epigenetic mechanisms are governing the observed circRNA expression changes. Together, we provide a map of circRNA expression in EpSCs and their differentiated counterparts and shed light on potential function and regulation of differentially expressed circRNAs.

Circular RNA expression is abundant and correlated to aggressiveness in early-stage bladder cancer.

The functions and biomarker potential of circular RNAs (circRNAs) in various cancer types are a rising field of study, as emerging evidence relates circRNAs to tumorigenesis. Here, we profiled the expression of circRNAs in 457 tumors from patients with non-muscle-invasive bladder cancer (NMIBC). We show that a set of highly expressed circRNAs have conserved core splice sites, are associated with Alu repeats, and enriched with Synonymous Constraint Elements as well as microRNA target sites. We identified 113 abundant circRNAs that are differentially expressed between high and low-risk tumor subtypes. Analysis of progression-free survival revealed 13 circRNAs, among them circHIPK3 and circCDYL, where expression correlated with progression independently of the linear transcript and the host gene. In summary, our results demonstrate that abundant circRNAs possess multiple biological features, distinguishing them from low-expressed circRNAs and non-circularized exons, and suggest that circRNAs might serve as a new class of prognostic biomarkers in NMIBC.