Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot

Abstract

Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45–55 of the DMD gene, might improve patients’ symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45–55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44–56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5′ splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing.

Application whole exome sequencing for the clinical molecular diagnosis of patients with Duchenne muscular dystrophy; identification of four novel nonsense mutations in four unrelated Chinese DMD patients

“…The major function of dystrophin protein is to stabilize the muscle cells and protect them from necrosis and damage (Suzuki et al, 2016). It also plays a significant role in linking the actin microfilament with the cell membrane by the help of several anchoring proteins (Takeshita et al, 2017).…”

Newly identified APN splice isoforms suggest novel splicing mechanisms may underlie circRNA circularization in moth

“…CircRNA have been extensively studied in recent years and may play specific roles in cellular physiology. Research on the correlation between circRNA formation and exon skipping provides a new perspective from which previously observed splicing events can be explained . Among the four HaAPNs screened, HaAPN1 and HaAPN3 exhibited different head‐to‐tail junctions (Figs ), whereas no amplification product was obtained for HaAPN2 and HaAPN5 with divergent primers.…”

“…The splicing reaction is catalysed by the spliceosome, which is a large, highly dynamic, multicomponent complex . The splicing code is extremely intricate, and recent studies on the correlation between circular RNA (circRNA) formation and exon skipping have provided a new perspective from which to investigate alternative splicing . CircRNA have long been considered by‐products of splicing errors, and they are rare and likely lack biological functions in organisms .…”

Roles of lncRNAs and circRNAs in regulating skeletal muscle development

“…100 Chicken circFGFR2 promotes myoblast proliferation and differentiation by sponging miR-133a-5p and miR-29b-1-5p. 106 In an embryonal subtype rhabdomyosarcoma-derived cell line, circ-ZNF609 knockdown induces a specific block at the G1-S transition, a strong decrease in p-Akt protein levels and a change in the pRb/Rb ratio. 102 circFGFR4 binding to miR-107 promotes cell differentiation by targeting Wnt3a in bovine primary myoblasts.…”

“…However, DMD circRNA biogenesis can lead to a shorter functional dystrophin protein, which could rescue the DMD phenotype into an asymptomatic or mild phenotype. 106 In an embryonal subtype rhabdomyosarcoma-derived cell line, circ-ZNF609 knockdown induces a specific block at the G1-S transition, a strong decrease in p-Akt protein levels and a change in the pRb/Rb ratio. Circ-ZNF609 acts by counteracting p-Akt proteasome-dependent degradation, thus acting as a newly identified regulator of the cell proliferation-related pathway.…”