Enzyme-free digital counting of endogenous circular RNA molecules in B-cell malignancies.

Circular RNAs (circRNAs) are covalently closed endogenous molecules with tissue- and disease-specific expression patterns, which have potential as diagnostic and prognostic biomarkers in cancer. The molecules are formed by a backsplicing event linking the 3'-end of an exon to the 5'-end of the same or an upstream exon, and they exert diverse regulatory functions important in carcinogenesis. The landscape of circRNA expression has not been characterized in B-cell malignancies, and current methods for circRNA quantification have several limitations that prevent development of clinically applicable assays. Here, we demonstrate that circRNAs can be accurately quantified without enzymatic reactions or bias using color-coded probes (NanoString technology). First, we performed high-throughput RNA sequencing (RNA-seq) of several mantle cell lymphoma and multiple myeloma cell lines to profile the genome-wide landscape of circRNA expression. We detected several circRNAs known to be deregulated in other cancers and identified a novel circRNA from the IKZF3 gene. Based on these data, we selected 52 unique circRNAs for which we designed color-coded probes spanning their specific backsplicing junctions. These circRNAs were quantified in cell lines and patient samples from several different B-cell malignancies (mantle cell lymphoma, multiple myeloma, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma and chronic lymphocytic leukemia) simultaneously using the NanoString technology. The circRNA expression profiles obtained could distinguish different B-cell malignancies, and confirmed the presence of the novel circRNA derived from IKZF3. The NanoString assays were specific for circRNA detection and data were more reproducible and quantitatively more accurate than RNA-seq data. In addition, we obtained high-quality data on severely degraded RNA samples from formalin-fixed, paraffin-embedded (FFPE) tissues. Together, we provide a map of circRNA expression in B-cell malignancies and present an enzyme-free digital counting methodology, which has the potential to become a new gold standard for circRNA quantification.

A Circular RNA Expressed from the FAT3 Locus Regulates Neural Development.

Circular RNAs (circRNAs) are key regulators of cellular processes, are abundant in the nervous system, and have putative regulatory roles during neural differentiation. However, the knowledge about circRNA functions in brain development is limited. Here, using RNA-sequencing, we show that circRNA levels increased substantially over the course of differentiation of human embryonic stem cells into rostral and caudal neural progenitor cells (NPCs), including three of the most abundant circRNAs, ciRS-7, circRMST, and circFAT3. Knockdown of circFAT3 during early neural differentiation resulted in minor transcriptional alterations in bulk RNA analysis. However, single-cell transcriptomics of 30 and 90 days differentiated cerebral organoids deficient in circFAT3 showed a loss of telencephalic radial glial cells and mature cortical neurons, respectively. Furthermore, non-telencephalic NPCs in cerebral organoids showed changes in the expression of genes involved in neural differentiation and migration, including FAT4, ERBB4, UNC5C, and DCC. In vivo depletion of circFat3 in mouse prefrontal cortex using in utero electroporation led to alterations in the positioning of the electroporated cells within the neocortex. Overall, these findings suggest a conserved role for circFAT3 in neural development involving the formation of anterior cell types, neuronal differentiation, or migration.