Analysis of Circular RNAs Identified from iPSC-Derived Cortical Neurons from Individuals with Idiopathic Autism

Background: Induced pluripotent stem cells (iPSCs) provide an attractive model of the cellular and molecular changes that underlie autism (AUT). Previous RNAseq analysis in our laboratory from cortical neurons derived from iPSC lines of individuals with idiopathic AUT and typically developing controls identified sets of differentially expressed genes enriched in pathways including neuronal differentiation, axon guidance, cell migration, and neural region patterning (De Rosa 2018). However, these protein coding RNAs represent only a fraction of the total transcripts identified and the non-coding repertoire, including circular RNAs (circRNAs), remains mostly unexplored. circRNAs are abundantly expressed, formed by alternative splicing of pre-mRNA, and have functions including post-transcriptional regulation, RNA binding protein sequestration, and transcriptional machinery scaffolding. These diverse functions suggest that differential expression of these molecules may have important developmental roles related to AUT.

Objectives: The goal of this study is to compare differentially regulated non-coding circRNAs in idiopathic AUT-specific iPSC-derived cortical neurons vs neurons derived from iPSCs from typically developing controls. Moreover, we aim to identify temporal regulation and differential expression of circRNAs by examining their profiles over a developmental time course in both AUT and control neurons.

Methods: iPSC lines were created from peripheral blood mononuclear cells (PBMCs) and developed from six individuals with autism and five typically developing controls. These iPSC lines were differentiated into cortical neurons and RNA was extracted at three time points post initiation of differentiation (day 35, 85, and 135). Total RNA was ribosomal RNA depleted and sequenced to at least 50 million paired end 100bp on the HiSeq2500. Following processing and alignment of reads to the hg19 reference genome with the TopHat2 aligner, three separate methods were used to detect circRNA in the samples: CIRI2, CIRCexplorer2, and circRNA_finder. Intronic and intergenic circRNA were discarded and exonic circRNAs with ≥ 3 junction spanning reads in ≥ 3 samples were considered expressed.

Results: In total, we identified ~11,000 expressed circRNA across all of the samples. Only 35% of circRNAs were identified by all three callers, but that number increased to over 70% when including only those with at least 15 junction reads. Individual sample counts rangd from 1200-8000 with the counts highly correlated with number of total reads (Pearson correlation coefficient = 0.86). There was no difference in overall circRNA counts between AUT and controls, nor were there differences between samples or groups across time points. Looking at specific circRNAs, we did identify the well characterized CDR1-AS, a circular RNA highly expressed in the central nervous system that functions as a sponge for miR-7 (Memczak, et. al., 2013, Nature). Moreover, we identified 445 circRNAs from 103 genes considered either syndromic or a strong candidate according to the SFARI Human Gene database. We are currently investigating the differential expression of these candidates.

Conclusions: The results of this study show that there is a diversity of circRNAs are expressed in cortical neurons derived from AUT-specific and control iPSC lines. Continued exploration of these and other noncoding transcripts will identify novel gene regulatory roles in autism.