The Brain’s Circular RNAs
Circular RNA (circRNA) has emerged as the latest large class of animal RNA. The circular structure creates a higher level of RNA stability, which has lead to the suspicion that circRNAs take on different functions than their linear counterparts. One pioneering example is the function as a miRNA sponge, where the circRNA ‘soaks up’ miRNAs meant to target linear transcripts. circRNAs are believed to come about by back-splicing reactions, where the 3’ end of one exon is covalently linked to the 5’ end of an upstream exon, which creates a different exon orientation than canonical (linear) splicing. There is also evidence suggesting that reverse complementary sequences are contained in introns flanking the exons that can be circularized. This appears to promote their biogenesis as after transcription RNA editing the introns comes into play and influences whether the repetitive elements can base-pair and circularize the RNA. Interestingly, neural genes often expresses circRNAs and circRNAs are highly enriched for in some neural tissues. Taking their earlier work to the next level, Rybak-Wolf et al. show that circRNAs are quite prevalent in the mammalian brain by combining both experimental and computational workflows that allowed them to analyze humans and mice.
The approach involved sequencing RNA from different brain regions, primary neurons, isolated synapses, and in other reference tissues. However, it also involved some extra considerations, such as depleting ribosomal RNA before sequencing and also advanced algorithms to distinguish the head to tail junctions of circRNAs from linear splicing events. This methodology, which was previously developed, also allows for an idea of the ratio of circRNA to linear RNA for a gene. The analysis showed that there are tens of thousands of putative circRNAs in the mammalian brain with humans showing a higher number than mice. Next, the team confirmed circularity using independent experimentation on mouse circRNAs by using the 3’ to 5’ exonuclease R and Northern blots for both isoforms, which confirmed their findings. A notable candidate was Rims2, which had 20-fold higher expression than its linear counterpart in the adult mouse brain but is only expressed at low levels in other tissues. This same event was conserved in humans. Also of interest was that most linear isoforms show broader expression profiles than their circular counterparts, the same gene can produce circRNAs that contain different exons, and that the ratio of circRNA to linear RNA is different in different tissues.
Diving deeper into the brain, the team found that circRNAs show differential expression in brain regions and layers in those brain regions with a high level of enrichment in the synapse. This lead the team to examine neurodevelopmental processes where it was found that circRNAs also show differential expression during neuronal differentiation. Finally, the team began an evolutionary analysis where they found that 4,522 of the 15,849 mouse circRNAs were conserved in humans and 19 candidates had their circularity validated in both species. Interestingly, there was conservation amongst the flanking introns and while at a much lower level frequency, they also confirmed conservation between mammalian circRNAs and those in flies. Also, by knocking down ADAR1, which edits RNA, they found that circRNA biogenesis is greatly increased, a pattern that follows with the naturally fluctuating ADAR1 levels.
Ultimately, the report provides supporting evidence for the existence of circRNAs while also delivering some much needed evidence to support that circRNAs aren’t a splicing artefact. These findings open the door for a new RNA species by finding strong evolutionary conservation and a high enrichment in the mammalian brain.
Rybak-Wolf A, Stottmeister C, Glažar P, Jens M, Pino N, Giusti S, Hanan M, Behm M, Bartok O, Ashwal-Fluss R, Herzog M, Schreyer L, Papavasileiou P, Ivanov A, Öhman M, Refojo D, Kadener S, & Rajewsky N (2015). Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed. Molecular cell, 58 (5), 870-85 PMID: 25921068
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