Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing
Spinal muscular atrophy is the leading genetic cause of infant mortality and is caused by homozygous loss of the SMN1 gene. We investigated global transcriptome changes in the spinal cord of inducible SMA mice. SMN depletion caused widespread retention of introns with weak splice sites or belonging to the minor (U12) class. We further demonstrated accumulation of DNA double strand breaks in the spinal cord of SMA mice and in human SMA cell culture models. DNA damage was partially rescued by suppressing the formation of R-loops, which accumulated over retained introns. We propose that instead of single gene effects, pervasive splicing defects caused by SMN deficiency trigger a global DNA damage and stress response, thus compromising motor neuron survival.
mRNA-seq: Total spinal cord from SMA and Control mice (rescue experiment) at d20 and d30; human SH-SY5Y cells expressing SMN or Control shRNA for 7d; human iPSC-derived motor neurons expressing SMN or Control shRNA for 5d; total spinal cord from SMA and Control mice (induction experiment) at d10, d20, and d30. DRIP-seq: SH-SY5Y cells expressing SMN or Control shRNA for 7d and immunoprecipitated with S9.6 antibody targeting RNA:DNA hybrids.