Scientists discover key mechanism for altered SMN2 gene processing

Findings of SMA research also show promise of treating cells with ASOs

Steve Bryson, PhD avatar

by Steve Bryson, PhD |

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Scientists have identified a key mechanism responsible for altered processing in the body’s SMN2 gene that, in people with spinal muscular atrophy (SMA), fails to make the needed SMN protein, new data show.

The discovery centers on heterogeneous nuclear ribonucleoprotein R (hnRNPR), part of a family of proteins that the researchers found are responsible for the alternative processing of SMN2. It leads to an inactive form of the SMN protein — but one that does not compensate for the loss of the SMN1 gene, the underlying cause of the rare genetic condition.

“In the present study, we report that hnRNPR regulates splicing of SMN-encoding genes,” the researchers wrote. Splicing is part of the process in which genes are translated into proteins.

Moreover, the new research also showed that treating cells with an antisense oligonucleotide, or ASO, reduced the activity of hnRNPR and enhanced the proper processes in SMN2.

The findings were detailed in “HnRNPR strongly represses splicing of a critical exon associated with spinal muscular atrophy through binding to an exonic AU-rich element,” a study published in the journal Molecular Genetics and Genomics.

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Investigating the mechanisms underlying SMA

Most cases of SMA are caused by mutations in the SMN1 gene, which result in little or no SMN protein production, and the progressive loss of nerve cells, called motor neurons, that control movement.

Cells also can carry a second, almost identical SMN2 gene that encodes for SMN. The number of SMN2 gene copies SMA patients have is generally associated with SMN production, with more copies linked to a milder SMA type.

Instructions to make proteins such as SMN are first copied from the gene — which is made of DNA — into pre-messenger RNA (pre-mRNA), a rough draft of the information that contains exons. Exons are segments that carry instructions for the protein, which are separated by other elements called introns.

Splicing is the process of removing introns, leaving only exons and a mature mRNA molecule, which is then translated, or decoded, into a protein — which essentially are the workhorses of a cell. With some genes, alternative splicing to remove or skip over specific exons is a natural process allowing a single gene to encode more than one protein.

Due to single changes in SMN2’s DNA sequence, however, alternative splicing excludes its exon number 7, producing a shorter version of the SMN that is degraded quickly and cannot fully compensate for SMN1 loss.

Preventing SMN2 exon 7 exclusion and increasing SMN production is the mechanism of action for two approved SMA disease-modifying therapies: Evrysdi (risdiplam) and Spinraza (nusinersen). Both were developed and approved in the last five years.

Nevertheless, according to researchers, “our understanding of SMN2 exon 7 splicing is still limited.”

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hnRNPR protein found play key role

hnRNPs are a family of proteins that bind to pre-mRNA and regulate splicing to mRNA. One of the members of this family, hnRNPR, which has been associated with SMA through its interaction with SMN, has yet to be characterized.

Now the researchers, from China, investigated hnRNP to learn more about how it works.

In cells, the overproduction of hnRNPR induced more exon 7 removals from SMN2, as well as from SMN1, compared with other proteins known to regulate splicing — namely hnRNPQ, Sam68, and SRSF10. hnRNPR robustly decreased exon 7 inclusion from 48% in controls to 1% for SMN2, and from 98% to 27% for SMN1.

Conversely, suppressing hnRNPR, but not other regulators, led to a significant increase in exon 7 in the SMN2 gene.

Introducing mutations in and around exon 7 of the pre-mRNA allowed the team to identify the region at the end of exon 7 that binds to hnRNPR. This region was rich in uracil (U) and adenine (A), two building blocks of mRNA. Complete deletion of this AU-rich segment mostly abolished hnRNPR’s ability to remove exon 7.

Sam68, another protein that suppresses splicing events, also was found to bind to the AU-rich element, but to a weaker degree than hnRNPR. Despite these findings, additional experiments suggested that hnRNPR and Sam68 repress splicing through distinct mechanisms.

The hnRNPR gene undergoes alternative splicing, which results in four forms of the hnRNPR protein, referred to as isoforms. One splice leads to a full-length protein (R633), while the other three produce shortened proteins (R636, R595, and R535).

R595 isoform, which itself lacks exon 5, had “no (SMN1) or much weaker inhibitory effect (SMN2) on exon 7 splicing compared with other isoforms,” the researchers wrote. This isoform also bound to the AU-rich element of SMN2 pre-mRNA.

Exon 7 inclusion in SMN2 was enhanced when cells were treated with an antisense oligonucleotide (ASO) specifically designed to facilitate the skipping of exon 5 in the hnRNPR gene to mimic the R595 isoform. ASOs are RNA-based molecules that can influence splicing events, similar to Spinraza.

“We identified a new mechanism that deteriorates SMN2 exon 7 splicing,” the researchers wrote.

While previous findings have shown that “hnRNPR is involved in SMA pathogenesis [development] at the protein level through interaction with SMN,” they noted, “our findings add another layer of functional regulation of a disease-associated protein by an essential hnRNP family member.”