Feeling out of the loop about all the different non-coding RNAs being described? Well hold on, because circular RNAs (circRNA) are about to throw you for a loop. Mammalian circRNAs are created by covalent closure of transcribed exons into loops. These circRNAs are highly expressed in specific brain regions, and conserved between mice and humans. They are much more stable than other RNA species, and thus may carry out unique biological functions. However, to date no functions for these RNAs have been mechanistically found in vivo.
One of the best studied circRNAs in Cdr1as. Cdr1as is a circularized long non-coding RNA not present in linear form and highly expressed in mammalian brain specifically. Human CDR1as has 70 binding sites for the microRNA miR-7, which regulates brain-specific genes. The binding sites are not perfect, meaning Cdr1as-miR-7 is likely not sliced by Ago2, which is part of the RNA-induced silencing complex. Therefore, it is suspected that CDR1as acts as a sponge for miR-7, reducing the number of free miR-7 molecules without degrading them. Cdr1as also has a binding site for miR-671 with almost full complementarity, suggesting mir-671 may function to target Cdr1as for some slicing and dicing that releases its miR-7 cargo.
Nikolaus Rajewsky and colleagues from the Max Delbrück Center for Molecular Medicine in Germany sought to characterize Cdr1as and test the hypothesis that it acts as a sponge for miR-7. They used CRISPR/Cas9 to develop a Cdr1as knock out mouse to assess downstream molecular, cellular, and behavioural changes. The team made us of numerous approaches to characterize the relationship between these ncRNAs in cerebellum, cortex, hippocampus, and olfactory bulb.
Here’s what happened:
- Using a modified crosslinking-Immunoprecipitation (CLIP) assay called AGO-CLIP, they found that Cdr1as was the top target for miR-7
- RNA fluorescence in situ hybridization (FISH) revealed that Cdr1as is highly expressed in neurons and not glia, and more expressed in excitatory than inhibitory neurons
- miR-7 was downregulated in Cdr1as knockout mice
- Immediate early genes targeted by miR-7 were upregulated in knockout mouse brain regions
- Knockout mice had double the amount of spontaneous synaptic vesicle release, which means there is deregulation of synaptic firing
- Cdr1as Knockout mice had reduced prepulse inhibition (PPI) of the startle response, characteristic of schizophrenia and psychosis
These data support the hypothesis that Cdr1as is important for the regulation of neurological processes via sequestering miR-7. Specifically, it acts to transport miR-7 to the synapses to regulate immediate early genes, controlling synaptic functioning. Unbound miR-7 would otherwise be targeted for degradation in the cytoplasm. Rajewsky shares that “Maybe we should think about Cdr1as not as a ‘sponge’ but as a ‘boat.’ It prevents its passengers from drowning and also moves on to new ports”. Numerous other circRNAs are expressed at high levels in the brain, suggesting a previously unknown layer of transcriptional regulation for neurotransmission and brain function.
Go sponge up all the details for your next voyage over at Science, August 2017