Abstract
Spinal Muscular Atrophy (SMA) is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The second gene copy, SMN2, produces some, but not enough, functional SMN protein. SMN is essential to assemble small nuclear ribonucleoproteins (snRNPs) that form the spliceosome. However, it is not clear whether SMA is caused by defects in this function that could lead to splicing changes in all tissues, or by the impairment of an additional, less well characterized, but motoneuron-specific SMN function. We addressed the first possibility by exon junction microarray analysis of motoneurons (MNs) isolated by laser capture microdissection from a severe SMA mouse model. This revealed changes in multiple U2-dependent splicing events. Moreover, splicing appeared to be more strongly affected in MNs than in other cells. By testing mutiple genes in a model of progressive SMN depletion in NB2a neuroblastoma cells, we obtained evidence that U2-dependent splicing changes occur earlier than U12-dependent ones. As several of these changes affect genes coding for splicing regulators, this may acerbate the splicing response induced by low SMN levels and induce secondary waves of splicing alterations.
Keywords:
ESE, exonic splicing enhancer; FCS, fetal calf serum; MN, motoneuron; NMD, nonsense-mediated mRNA decay; NMJ, neuromuscular junction, PCR; RT, reverse transcription; SMA, Spinal Muscular Atrophy; SMN, Survival Motor Neuron; Spinal Muscular Atrophy; TcRβ, T-cell receptor β chain; exon junction microarray; hz, heterozygote, LCM; laser capture microdissection; major spliceosome; minor spliceosome; motoneurons; neurodegerative disease; polymerase chain reaction, qPCR; real-time (quantitative) PCR; sh, short hairpin; snRNA, small nuclear ribonucleic acid; snRNP assembly; snRNP, small nuclear ribonucleoprotein; splicing; splicing regulators.