Sequence and expression levels of circular RNAs in progenitor cell types during mouse corticogenesis

Abstract

Circular (circ) RNAs have recently emerged as a novel class of transcripts whose identification and function remain elusive. Among many tissues and species, the mammalian brain is the organ in which circRNAs are more abundant and first evidence of their functional significance started to emerge. Yet, even within this well-studied organ, annotation of circRNAs remains fragmentary, their sequence is unknown, and their expression in specific cell types was never investigated. Overcoming these limitations, here we provide the first comprehensive identification of circRNAs and assessment of their expression patterns in proliferating neural stem cells, neurogenic progenitors, and newborn neurons of the developing mouse cortex. Extending the current knowledge about the diversity of this class of transcripts by the identification of nearly 4,000 new circRNAs, our study is the first to provide the full sequence information and expression patterns of circRNAs in cell types representing the lineage of neurogenic commitment. We further exploited our data by evaluating the coding potential, evolutionary conservation, and biogenesis of circRNAs that we found to arise from a specific subclass of linear mRNAs. Our study provides the arising field of circRNA biology with a powerful new resource to address the complexity and potential biological significance of this new class of transcripts.

microRNA profiling of mouse cortical progenitors and neurons reveals miR-486-5p as a novel regulator of neurogenesis

“…log2 fold change ≥ 0.58 or ≤ −0.58, respectively, FDR <5%) in one cell type compared to its parental population. As observed previously for linear and circular transcripts (31,35), only a small fraction of miRNAs showed a significant change between PP-DP (7%) and DP-N (17%) while the majority of those up- or down-regulated between PP-DP continued to follow the same trend of up- or down-regulation, respectively, between DP-N ( Figure 2 ).…”

“…Aiming to profile global miRNA expression during cortical development, we isolated PP, DP and N (each in three biological replicates) from the lateral cortices of Btg2::RFP/Tubb3::GFP mouse embryos at E14.5, as previously described (31,35) ( Figure 1a ). Total RNA was used for cDNA library preparation and small RNAs were isolated by size selection, followed by 75-bp high-throughput sequencing.…”

“…Here we exploited the Btg2::RFP/Tubb3::GFP mouse line as a well-established tool used by our group in previous studies to characterize the molecular signature of neurogenic commitment (34-37). By doing so, our group identified switch transcripts belonging to several classes of RNAs and including coding, long non-coding and circular RNAs and in most cases showing their functional roles in brain development (31,35,36). Continuing this line of research, here not only we provided the field with a validated and overall complete catalogue of cortical miRNAs at single-population level but also identified in miR-486a/b-5p a novel regulator of neurogenesis.…”

“…Cells were resuspended in 1 ml of ice-cold PBS and 10 µl of 7-AAD (BD Pharmingen) were added for dead cells discrimination. Sorting was performed by BD FACSAria™ III (BD Biosciences) with previously described gating (31,35). A minimum of 1 × 10 6 cells per sample was collected in PBS and centrifuged (300 g, 10 min at 4°C) before RNA extraction.…”

“…Validation and use of this mouse line revealed to be very powerful in the identification of several new genes and biological processes regulating cortical development (31). This included the thorough characterization of the elusive class of long non-coding (34) and circular (35) RNAs, novel transcription factors involved in corticogenesis (36) and a comprehensive description of DNA methylation and hydroxymethylation as epigenetic marks tuning brain development (37).…”