Defective minor spliceosomes induce SMA-associated phenotypes through sensitive intron-containing neural genes in Drosophila

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.

Regulation of Membrane Phospholipid Homeostasis in Neurodegenerative Diseases

“…In addition, Siberian tiger Pcyt2 transcript (ENSPTIG00000010590) lacks the C-domain HXGH, gorilla variant (ENSGGOG00000067604.1) has the N-domain HXGH absent; zebrafish has several variants lacking either of the two HXGH catalytic sites. Excitingly, the most recent study in Drosophila established that dysfunctional minor spliceosome causes spinal muscular atrophy by direct contribution from three neural genes, Pcyt2, Zmynd10, and Fas3 as the main splicing targets [73]. This agreed with the existence of multiple Pcyt2 isoforms and implicates a strong splicing and self-inhibiting regulation of de novo PE synthesis by the Kennedy pathway.…”

Two oppositely-charged sf3b1 mutations cause defective development, impaired immune response, and aberrant selection of intronic branch sites in Drosophila

“…Alternative splicing is the consequence of competition/selection between multiple SSs. To address details of SS selection, we used our recently developed tool ΔUSS ( D ifferential U sage of S plice S ite) to evaluate all the individual SSs in the Drosophila transcriptome [ 74 ] ( Fig 6A ). In total, usages of 417 and 472 of 5′SSs, and 404 and 524 of 3′SSs were significantly changed in H698D and H698R , respectively ( Fig 6B and S6 Table ).…”

“…The wild type ( WT ) Drosophila melanogaster used in this study is a w1118 isogenic strain (BDSC 5905). Point mutant strains were constructed using the CRISPR/Cas9 system [ 74 ]. In brief, the target sequence of each guide RNA (sgRNA) was selected, donor plasmids with point mutations and the adjacent 3 kb sequences as homologous arms were constructed using pMD18-T ( Fig 1B ), and the gRNA and donor plasmids were co-injected into embryos of the transgenic line nanos-Cas9 by UniHuaii Technology Company.…”

“…Significant DS events were screened by conditions |ΔPSI| > 0.05 and FDR < 0.05. Differential splice site usage was analyzed by ΔUSS, which is modified from an Unused Index as described [ 74 ]. Significant ΔUSS were screened by conditions |ΔUSS| > 0.01, p value <0.05.…”

Comprehensive Identification and Alternative Splicing of Microexons in Drosophila

“…Ribonucleic acid samples were prepared as described ( Li et al, 2020 ), and the construction of cDNA libraries and sequencing were performed using Illumina Hi-Seq 2000 ( Stark et al, 2019 ). The head, body, and gonads of the female and male Drosophila were sequenced in the form of 150 bp fr-firststrand pair-end reads, and the embryo, 3L larvae, and adults were sequenced in the form of 100 bp fr-untstranded pair-end reads.…”