Researchers have created stable cell lines that are able to express circular RNAs (circRNAs) created from the gene responsible for spinal muscular atrophy (SMA), according to an article published in Genes.
Mutations in the SMN1 gene, which encodes the survival motor neuron (SMN) protein, result in SMA. A second gene, SMN2, also produces SMN but at a much lower rate due to predominant skipping of exon 7, leading to a truncated and unstable molecule.
In addition to SMN protein, both genes also produce circRNAs containing 2 or more exons. These circRNAs are usually generated by the first 4 internal exons (2A, 2B, 3, and 4), and the most common circRNAs include C2A-2B-3-4, C2B-3-4, and C3-4, which contain exons 3 and 4 with or without 2A and 2B.
Not much was known previously about the mechanism of creating circRNAs from the SMN protein, but researchers in the current study were able to reliably express circRNAs in both HeLa cells and inducible HEK293 cells.
In the HeLa cells, an engineered plasmid vector pC2B-3-4 was used to generate C2B-3-4 circRNAs. The researchers were also able to use similar methods to produce circRNAs from nonmammalian eGFP and human HIPK2 exon 2. The expression of C2B-3-4 increased more than 2-fold in the HeLa cells when the researchers included introns in the pC2B-2-3 vector.
Read more about SMA etiology
A stable cell line was also produced during the study by integrating a single transgene into the DNA of HEK293 cells. Through colony selection, a cell line was chosen that produced C2B-3-4 at levels comparable to transiently transfected cells at the highest plasmid levels. Through analysis of nuclear and cytoplasmic samples from the stable cell lines, it was found that the overexpression of the SMN circRNA was localized to the cytoplasm.
“These findings provide new insights regarding our understanding of circRNA generation and open avenues to uncover novel functions of the SMN genes,” the authors said.
Previous studies have shown that the SMN genes can produce a large number of different circRNAs, which are categorized into 4 main types: those that harbor early exons and use the 5’ss of exon 4 or upstream exons (type 1); those that harbor both upstream and downstream exons (type 2); those that use the 5’ss located downstream of exon 7 (type 3); and those formed by trans-splicing with 1 or more exons from other genes (type 4).
Luo D, Singh NN, Singh RN. Internal introns promote backsplicing to generate circular RNAs from spinal muscular atrophy gene. Genes. 2022;13(7):1145. doi:10.3390/genes13071145