Functional Characterisation of the Circular RNA, circHTT(2-6), in Huntington’s Disease

Abstract

Trinucleotide repeat disorders comprise ~20 severe, inherited, human neuromuscular and neurodegenerative disorders, which result from an abnormal expansion of repetitive sequences in the DNA. The most common of these, Huntington’s disease (HD), results from expansion of the CAG repeat region in exon 1 of the HTT gene via an unknown mechanism. Since non-coding RNAs have been implicated in the initiation and progression of many diseases, herein we focused on a circular RNA (circRNA) molecule arising from non-canonical splicing (backsplicing) of HTT pre-mRNA. The most abundant circRNA from HTT, circHTT(2-6), was found to be more highly expressed in the frontal cortex of HD patients, compared with healthy controls, and positively correlated with CAG repeat tract length. Furthermore, the mouse orthologue (mmu_circHTT(2-6)) was found to be enriched within the brain and specifically the striatum, a region enriched for medium spiny neurons that are preferentially lost in HD. Transgenic overexpression of circHTT(2-6) in two human cell lines—SH-SY5Y and HEK293—reduced cell proliferation and nuclear size without affecting cell cycle progression or cellular size, or altering the CAG repeat region length within HTT. CircHTT(2-6) overexpression did not alter total HTT protein levels, but reduced its nuclear localisation. As these phenotypic and genotypic changes resemble those observed in HD patients, our results suggest that circHTT(2-6) may play a functional role in the pathophysiology of this disease.

Regulatory mechanism of circular RNAs in neurodegenerative diseases

“…Another study found that circHTT 2–6 derived from the exons 2, 3, 4, 5, and 6 of the HTT gene was enriched in in the frontal cortex of HD patients 92 . When circHTT (2–6) was overexpressed in HEK293 and SH‐SY5Y cells, no change in the CAG repeat region of HD was detected, which decreased the cell proliferation, nuclear area, and altered subcellular localization of the HTT protein 92 . These results demonstrate the overexpression of circHTT undergoes HD‐related pathological changes, but its specific functional mechanism needs further study.…”

Versatile toolkit for highly-efficient and scarless overexpression of circular RNAs

“…To assess the CircRNA mini vector, hereafter called mc, six circRNAs were restriction enzyme-cloned into this vector -EcoRV/SacII cloning sites: circEGFR (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), circEGFR (2)(3)(4)(5)(6), circEGFR (19)(20), circTNIK (5)(6)(7); HpaI/AgeI cloning sites: circNFASC (29)(30), and using the AgeI site only: circBCR(9-14) (Figures 1A-B). All plasmid sequences were confirmed by Sanger sequencing.…”

“…To more thoroughly investigate the fidelity of the circRNA bsj sequence produced from mc2 in cells transfected with this plasmid, a next generation sequencing strategy was devised for each individual circRNA, called circRNA bsj-seq. This was performed on five different circRNAs, including circBCR (9)(10)(11)(12)(13)(14) cloned into both mc2 and the original mc vector in addition to another four circRNAs: circEGFR (19,20), circEGFR (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), circMLL (9) and circMLLT2 (3,4). Using a 12-mer unique molecular identifier, at least 185,000 individual circRNAs were sequenced from HEK293 cells transfected with each different mc/mc2 construct.…”

“…CircRNAs formed in this manner are a theoretical possibility for any gene with multiple exons, yet not all transcribed, spliced genes produce circRNAs (4). Indeed, the expression of circRNA is dynamic and tightly regulated, with some conserved across species, and their expression can be independent of the parental mRNA (5)(6)(7). This ancient and conserved gene expression phenomena implies functions for circRNAs.…”

“…This native milieu was modified to augment circularisation efficiency by including a copy of the 5' intron region (typically 100-750 nucleotides in length) downstream of the circRNA exon in a reverse complement orientation, thereby producing a long hairpin structure between the introns that promotes circularisation of the intervening exons. This approach has yielded high fidelity circRNA overexpression in most instances, including our own laboratory, but this strategy demands multiple PCR, restriction digestion, ligation and sequencing verification steps (7,20,21). Furthermore, due to the long reverse complementary stretch of the amplicon, it is not amenable to high-throughput cloning strategies, including use of synthetic gene fragments.…”