Scientists create SMN super minigene as tool for studying SMA

It may help in modeling disease-causing mutations and studying their impact

Steve Bryson, PhD avatar

by Steve Bryson, PhD |

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A close-up view of a strand of DNA highlights its double-helix structure.

A team of scientists in Iowa have created a super minigene  — a small gene fragment used as a tool to study genetic manipulation — that recapitulates essential features of the natural SMN1 and SMN2 genes linked to spinal muscular atrophy (SMA) and its severity.

One of the most important applications for this new super minigene, the researchers noted, could be the modeling of various disease-causing mutations to investigate their impact on biological processes in different cell types.

“Once such mutations are introduced in the super minigene, their effects could be monitored at multiple levels,” the researchers wrote.

It also may be used to screen for therapeutic compounds that enhance SMN protein production, which is deficient in people with SMA, according to the team.

Their study, “A super minigene with a short promoter and truncated introns recapitulates essential features of transcription and splicing regulation of the SMN1 and SMN2 genes,” was published in the journal Nucleic Acids Research.

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An illustration showing a pair of scissors splicing into the DNA helix.

Gene editing of SMN2 leads to normal SMN protein levels: Study

Super minigene recaps features of SMN1 and SMN2 genes

Mutations in the SMN1 gene cause most cases of SMA, a disorder marked by progressive muscle weakness and wasting, known as atrophy. Such mutations result in little or no SMN protein, leading to the progressive loss of motor neurons, the nerve cells that control movement.

Cells also have a second SMN2 gene that encodes SMN protein and is almost identical to SMN1. Generally, the number of SMN2 gene copies is associated with some SMN production, and patients with more copies have a milder type of SMA.

All genes that encode proteins have so-called exons and introns along the DNA strand: exons carry instructions for the protein and introns do not.

In the first step of protein production, an enzyme called RNA polymerase copies, or transcribes, the information from the gene into pre-messenger RNA, essentially creating a rough draft. Then, in a process called splicing, the introns are removed to form a mature messenger RNA, or mRNA, which is then read, or translated, by the cell’s machinery to generate a protein.

Alternate splicing is a natural process in which some exons are excluded to allow a single gene to encode more than one protein. However, due to single changes in SMN2’s DNA sequence, alternative splicing excludes exon number 7, producing a shorter version of the SMN protein that is degraded quickly. As such, it cannot fully compensate for SMN1 loss.

Two approved SMA disease-modifying therapies — Spinraza (nusinersen) and Evrysdi (risdiplam) — work by preventing SMN2 exon 7 exclusion, thus increasing SMN production.

To understand the details of SMN2 expression, or activity, and associated splicing events, researchers at Iowa State University created an SMN2 super minigene on a circular strand of DNA that recapitulates all of the essential features of the natural SMN2 gene.

This super minigene carried the exons that contain SMN protein information, namely 1, 2A, 2B, 3, 4, 5, 6, 7 and 8. It also had shorter intron segments, but the DNA sequences next to the exons exactly matched those from natural SMN2. The minigene retained the promotor, a segment in front of exon 1 where RNA polymerase binds.

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A close-up view of a strand of DNA highlights its double-helix structure.

Scientists discover key mechanism for altered SMN2 gene processing

Researchers call new tool ‘unique, powerful’

The team then inserted the super minigene into human cells to examine RNA expression and splicing patterns generated from SMN2.

The results showed that the SMN2 super minigene produced full-length mRNA as the predominant product, followed by an mRNA without exon 7, as seen in SMA patients. The third most abundant mRNA had both exons 5 and 7 missing. The researchers confirmed that the minigene was able to produce SMN protein.

Experiments showed that removing DNA segments from the promotor region of the minigene appeared to have no effect on splicing. Still, some segments were critical to the level of gene expression. Also, overexpressing SMN protein was found to trigger the exclusion of exon 3 in natural SMN2 as well as SMN1, which affected downstream splicing events.

The splicing of multiple exons in both the SMN2 minigene and natural SMN1 and SMN2 was found to be regulated by two proteins, SRSF3 and DHX9. Moreover, the expression of the SMN2 minigene lacking exon 7, compared with full-length SMN2, led to alternative splicing events for natural SMN1 and SMN2.

The super minigene we report here represents a unique, powerful and easily amenable tool for discovering regulatory elements associated with several aspects of RNA metabolism as well as for therapeutic and diagnostic applications.

Researchers then validated the super minigene as a reporter system to monitor SMN protein levels upon splicing correction by SMA disease-modifying therapies. They inserted the minigene into cells with a molecule almost identical to nusinersen, the active ingredient in Spinraza. The levels of SMN protein were significantly increased compared with a control molecule.

Finally, a super minigene was created to recapitulate the splicing patterns of the natural SMN1 that carried a mutation associated with a severe form of SMA. This mutation is located in intron 7, which leads to the exclusion of exon 7 in SMN1. As expected, they observed complete skipping of exon 7 and the retention of intron 7 in all cell types tested. However, additional splicing patterns varied depending on the type of cell used.

“Our findings are significant given the fact that no information on tissue-specific splicing of this pathogenic [disease-causing] mutation is currently available,” the researchers wrote.

“The super minigene we report here represents a unique, powerful and easily amenable tool for discovering regulatory elements associated with several aspects of RNA metabolism as well as for therapeutic and diagnostic applications,” they concluded.