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Abstract
The minor spliceosome is evolutionarily conserved in higher eukaryotes, but its biological significance remains poorly understood. Here, by precise CRISPR/Cas9-mediated disruption of the U12 and U6atac snRNAs, we report that a defective minor spliceosome is responsible for spinal muscular atrophy (SMA) associated phenotypes in Drosophila. Using a newly developed bioinformatic approach, we identified a large set of minor spliceosome-sensitive splicing events and demonstrate that three sensitive intron-containing neural genes, Pcyt2, Zmynd10, and Fas3, directly contribute to disease development as evidenced by the ability of their cDNAs to rescue the SMA-associated phenotypes in muscle development, neuromuscular junctions, and locomotion. Interestingly, many splice sites in sensitive introns are recognizable by both minor and major spliceosomes, suggesting a new mechanism of splicing regulation through competition between minor and major spliceosomes. These findings reveal a vital contribution of the minor spliceosome to SMA and to regulated splicing in animals.
Highlights
The minor spliceosome is evolutionarily conserved in higher eukaryotes, but its biological significance remains poorly understood
The heterozygous strains (U12+/Δ and U6atac+/Δ) appeared to grow normally; the homozygous strains are lethal at pupal stages (Fig. 1c), indicating that small nuclear RNAs (snRNAs) components of the minor spliceosome are essential for fly development
To examine if the rescue effect of neuromuscular junction (NMJ) by the four genes was specific to SMN ablation, we expressed these genes in WT Drosophila and observed that neural-specific expression of CG10171 led to the increase of the NMJ boutons, whereas Zmynd[10], Pcyt[2] or Fas[3] had no effect (Supplementary Fig. 5b, c). These results demonstrated that the restoration of neuromuscular connections in SmnΔ/Δ by the expression of Zmynd[10], Pcyt[2], and Fas[3] are reliable, and CG10171 may work on certain SMN independent pathway to regulate NMJ boutons
Summary
The minor spliceosome is evolutionarily conserved in higher eukaryotes, but its biological significance remains poorly understood. Many splice sites in sensitive introns are recognizable by both minor and major spliceosomes, suggesting a new mechanism of splicing regulation through competition between minor and major spliceosomes. These findings reveal a vital contribution of the minor spliceosome to SMA and to regulated splicing in animals. In SMA models, deficiency of SMN protein results in altered stoichiometry of both the major and minor snRNAs25,31,32 and widespread changes of pre-mRNA splicing[33,34,35,36]. Above mentioned mutations in the human minor-spliceosomal components do not cause SMA, but rather cause other diseases that have completely different phenotypes from SMA
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