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The expression profiles and spatial distributions of RNAs regulate many cellular functions. Image-based transcriptomic approaches provide powerful means to measure both expression and spatial information of RNAs in individual cells within their native environment. Among these approaches, multiplexed error-robust fluorescence in situ hybridization (MERFISH) has achieved spatially resolved RNA quantification at transcriptome scale by massively multiplexing single-molecule FISH measurements. Here, we increased the gene throughput of MERFISH and demonstrated simultaneous measurements of RNA transcripts from ∼10,000 genes in individual cells with ∼80% detection efficiency and ∼4% misidentification rate. We combined MERFISH with cellular structure imaging to determine subcellular compartmentalization of RNAs. We validated this approach by showing enrichment of secretome transcripts at the endoplasmic reticulum, and further revealed enrichment of long noncoding RNAs, RNAs with retained introns, and a subgroup of protein-coding mRNAs in the cell nucleus. Leveraging spatially resolved RNA profiling, we developed an approach to determine RNA velocity in situ using the balance of nuclear versus cytoplasmic RNA counts. We applied this approach to infer pseudotime ordering of cells and identified cells at different cell-cycle states, revealing ∼1,600 genes with putative cell cycle-dependent expression and a gradual transcription profile change as cells progress through cell-cycle stages. Our analysis further revealed cell cycle-dependent and cell cycle-independent spatial heterogeneity of transcriptionally distinct cells. We envision that the ability to perform spatially resolved, genome-wide RNA profiling with high detection efficiency and accuracy by MERFISH could help address a wide array of questions ranging from the regulation of gene expression in cells to the development of cell fate and organization in tissues.

Since decades it has been known that non-protein-coding RNAs have important cellular functions. Deep sequencing recently facilitated the discovery of thousands of novel transcripts, now classified as long noncoding RNAs (lncRNAs), in many vertebrate and invertebrate species. LncRNAs are involved in a wide range of cellular mechanisms, from almost all aspects of gene expression to protein translation and stability. Recent findings implicate lncRNAs as key players of cellular differentiation, cell lineage choice, organogenesis and tissue homeostasis. Moreover, lncRNAs are involved in pathological conditions such as cancer and cardiovascular disease, and therefore provide novel biomarkers and pharmaceutical targets. Here we discuss examples illustrating the versatility of lncRNAs in gene control, development and differentiation, as well as in human disease.

Background Several of the thousands of human long noncoding RNAs (lncRNAs) have been functionally characterized, yet their potential involvement in hepatocellular carcinoma (HCC) remains poorly understood. Methods LncRNA-HOXD-AS1 was identified by microarray and validated by real-time PCR. The clinicopathological significance of HOXD-AS1 was analyzed by Kaplan-Meier method. Chromatin immunoprecipitation was conducted to examine the mechanism of HOXD-AS1 upregulation. The role of HOXD-AS1 in HCC cells was assessed both in vitro and in vivo. ceRNA function of HOXD-AS1 was evaluated by RNA immunoprecipitation and biotin-coupled miRNA pull down assays. Results In this study, we found that HOXD-AS1 was significantly upregulated in HCC tissues. Clinical investigation demonstrated high expression level of HOXD-AS1 was associated with poor prognosis and high tumor node metastasis stage of HCC patients, and was an independent risk factor for survival. Moreover, our results revealed that STAT3 could specifically interact with the promoter of HOXD-AS1 and activate HOXD-AS1 transcription. Knockdown of HOXD-AS1 significantly inhibited migration and invasion of HCC cells in vitro and distant lung metastasis in vivo. Additionally, HOXD-AS1 was enriched in the cytoplasm, and shared miRNA response elements with SOX4. Overexpression of HOXD-AS1 competitively bound to miR-130a-3p that prevented SOX4 from miRNA-mediated degradation, thus activated the expression of EZH2 and MMP2 and facilitated HCC metastasis. Conclusions In summary, HOXD-AS1 is a prognostic marker for HCC patients and it may play a pro-metastatic role in hepatocarcinogenesis.

Although many long noncoding RNAs (lncRNAs) have been identified in human and other mammalian genomes, there has been limited systematic functional characterization of these elements. In particular, the contribution of lncRNAs to organ development remains largely unexplored. Here we analyse the expression patterns of lncRNAs across developmental time points in seven major organs, from early organogenesis to adulthood, in seven species (human, rhesus macaque, mouse, rat, rabbit, opossum and chicken). Our analyses identified approximately 15,000 to 35,000 candidate lncRNAs in each species, most of which show species specificity. We characterized the expression patterns of lncRNAs across developmental stages, and found many with dynamic expression patterns across time that show signatures of enrichment for functionality. During development, there is a transition from broadly expressed and conserved lncRNAs towards an increasing number of lineage- and organ-specific lncRNAs. Our study provides a resource of candidate lncRNAs and their patterns of expression and evolutionary conservation across mammalian organ development. A transcriptome dataset from seven organs and seven mammalian species throughout development is used to analyse the expression of long noncoding RNAs in tissues within and between species, and at different stages of organ development.

A long intergenic noncoding RNA, Firre, is now shown to localize to a domain across its own chromosomal locus and to distinct interacting transchromosomal loci in mouse and human cells. In addition, Firre interacts with nuclear-matrix factor hnRNPU. These results lead to a model in which Firre functions as a nuclear-organization factor modulating the topological organization of multiple chromosomes. RNA, including long noncoding RNA (lncRNA), is known to be an abundant and important structural component of the nuclear matrix. However, the molecular identities, functional roles and localization dynamics of lncRNAs that influence nuclear architecture remain poorly understood. Here, we describe one lncRNA, Firre, that interacts with the nuclear-matrix factor hnRNPU through a 156-bp repeating sequence and localizes across an ~5-Mb domain on the X chromosome. We further observed Firre localization across five distinct trans -chromosomal loci, which reside in spatial proximity to the Firre genomic locus on the X chromosome. Both genetic deletion of the Firre locus and knockdown of hnRNPU resulted in loss of colocalization of these trans -chromosomal interacting loci. Thus, our data suggest a model in which lncRNAs such as Firre can interface with and modulate nuclear architecture across chromosomes.

By integrating the expression profiles of long noncoding RNAs (lncRNAs) with clinical outcome and somatic copy-number alteration, the authors identified new lncRNAs that are associated with certain cancer subtypes and clinical prognoses. Experimental validation of the prostate cancer cell growth dependence of two new lncRNAs demonstrates the power of this approach for discovering disease-related lncRNAs. Despite growing appreciation of the importance of long noncoding RNAs (lncRNAs) in normal physiology and disease, our knowledge of cancer-related lncRNAs remains limited. By repurposing microarray probes, we constructed expression profiles of 10,207 lncRNA genes in approximately 1,300 tumors over four different cancer types. Through integrative analysis of the lncRNA expression profiles with clinical outcome and somatic copy-number alterations, we identified lncRNAs that are associated with cancer subtypes and clinical prognosis and predicted those that are potential drivers of cancer progression. We validated our predictions by experimentally confirming prostate cancer cell growth dependence on two newly identified lncRNAs. Our analysis provides a resource of clinically relevant lncRNAs for the development of lncRNA biomarkers and the identification of lncRNA therapeutic targets. It also demonstrates the power of integrating publically available genomic data sets and clinical information for discovering disease-associated lncRNAs.

Noncoding RNAs (ncRNAs) play prominent roles in the regulation of gene expression via their interactions with other biological molecules such as proteins and nucleic acids. Although much of our knowledge about how these ncRNAs operate in different biological processes has been obtained from experimental findings, computational biology can also clearly substantially boost this knowledge by suggesting possible novel interactions of these ncRNAs with other molecules. Computational predictions are thus used as an alternative source of new insights through a process of mutual enrichment because the information obtained through experiments continuously feeds through into computational methods. The results of these predictions in turn shed light on possible interactions that are subsequently validated experimentally. This review describes the latest advances in databases, bioinformatic tools, and new in silico strategies that allow the establishment or prediction of biological interactions of ncRNAs, particularly miRNAs and lncRNAs. The ncRNA species described in this work have a special emphasis on those found in humans, but information on ncRNA of other species is also included.

Objectives Long non‐coding RNAs (lncRNAs) have been demonstrated as crucial regulators in cancer, but whether they are involved in the immune response of cancer cells remains largely undiscovered. GATA3‐AS1 is a novel lncRNA that was upregulated in breast cancer (BC) according to online databases. However, its role in triple‐negative breast cancer (TNBC) was elusive. Methods GATA3‐AS1 expression in BC tissues and adjacent normal tissues was obtained from online databases. Loss‐of‐function assays were designed and conducted to verify the functional role of GATA3‐AS1 in TNBC cells. Bioinformatic analysis and mechanism experiments were applied to explore the downstream molecular mechanism of GATA3‐AS1. Similarly, the upstream mechanism which led to the upregulation of GATA3‐AS1 in TNBC cells was also investigated. Results GATA3‐AS1 was markedly overexpressed in TNBC tissues and cells. Knockdown of GATA3‐AS1 suppressed TNBC cell growth and enhanced the resistance of TNBC cells to immune response. GATA3‐AS1 induced the deubiquitination of PD‐L1 through miR‐676‐3p/COPS5 axis. GATA3‐AS1 destabilized GATA3 protein by promoting GATA3 ubiquitination. Conclusion GATA3‐AS1 contributed to TNBC progression and immune evasion through stabilizing PD‐L1 protein and degrading GATA3 protein, offering a new target for the treatment of TNBC.

Ferroptosis is a newly defined form of programmed cell death that plays an important role in many cancers. However, ferroptosis-related lncRNAs (FRLs) involved in the regulation of colon cancer are not thoroughly understood. This study aimed to identify a prognostic FRL signature in colon cancer and explore its potential molecular function. RNA-seq data and relevant clinical information were obtained from The Cancer Genome Atlas (TCGA) database, and a list of ferroptosis-related genes was extracted from the FerrDb website. Analysis of differentially expressed FRLs was performed using the 'limma' package in R software. By implementing coexpression analysis and univariate Cox analysis, we then identified prognostic FRLs. Using Cox regression analysis with the least absolute shrinkage and selection operator (LASSO) algorithm, we constructed a prognostic model based on 4 FRLs. We evaluated the prognostic power of this model using Kaplan-Meier (K-M) survival curve analysis and receiver operating characteristic (ROC) curve analysis. Moreover, the relationships between the signature and immune landscape, somatic mutation and drug sensitivity were explored. Finally, experiments were conducted to validate the functions of AP003555.1 and AC000584.1. A 4-FRL signature was constructed. Two risk groups were classified based on the risk score calculated by this signature. The signature-based risk score exhibited a more powerful capacity for survival prediction than traditional clinicopathological features in colon patients. Additionally, we observed a significant difference in immune cells, such as CD4+ and CD8+ T cells and macrophages, between the two groups. Moreover, the high-risk group exhibited lower IC50 values for certain chemotherapy drugs, such as cisplatin, docetaxel, bleomycin or axitinib. Finally, the experiments showed that ferroptosis processes were suppressed after AP003555.1 and AC000584.1 knockdown. The proposed 4-FRL signature is a promising biomarker to predict clinical outcomes and therapeutic responses in colon cancer patients.

Background Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, and tumor recurrence and metastasis are major factors that contribute to the poor outcome of patients with HCC. Long noncoding RNAs (lncRNAs) are known to regulate different tumorigenic processes, and a growing body of evidence indicates that Hippo kinase signaling is inactivated in many cancers. However, the upstream lncRNA regulators of Hippo kinase signaling in HCC are poorly understood. Methods Using a lncRNA microarray, we identified a novel lncRNA, uc.134, whose expression was significantly decreased in the highly aggressive HCC cell line HCCLM3 compared with MHCC97L cells. Furthermore, we evaluated uc.134 expression in clinical samples using in situ hybridization (ISH) and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The full-length transcript of uc.134 was confirmed using rapid amplification of cDNA ends (RACE) analyses. To investigate the biological function of uc.134, we performed gain-of-function and loss-of-function studies both in vitro and in vivo. The underlying mechanisms of uc.134 in HCC were investigated using RNA pulldown, RNA immunoprecipitation, ubiquitination assays, Western blotting, mRNA microarray analyses, and qRT-PCR analyses. Results The ISH assay revealed that uc.134 expression was significantly decreased in 170 paraffin-embedded samples from patients with HCC compared with adjacent tissues and uc.134 expression directly correlated with patient prognosis. Furthermore, we defined a 1867-bp full-length transcript of uc.134 using 5′- and 3′-RACE analysis. The overexpression of uc.134 inhibited HCC cell proliferation, invasion, and metastasis in vitro and in vivo, whereas the knockdown of uc.134 produced the opposite results. Furthermore, we confirmed that uc.134 (1408–1867 nt) binds to CUL4A (592–759 aa region) and inhibits its nuclear export. Moreover, we demonstrated that uc.134 inhibits the CUL4A-mediated ubiquitination of LATS1 and increases YAP S127 phosphorylation to silence the target genes of YAP. Finally, a positive correlation between uc.134, LATS1, and pYAP S127 was confirmed in 90 paraffin-embedded samples by ISH and immunohistochemical staining. Conclusions Our study identifies that a novel lncRNA, uc.134, represses hepatocellular carcinoma progression by inhibiting the CUL4A-mediated ubiquitination of LATS1 and increasing YAP S127 phosphorylation. The use of this lncRNA may offer a promising treatment approach by inhibiting YAP and activating Hippo kinase signaling.