Splicing changes in SMA mouse motoneurons and SMN-depleted neuroblastoma cells: Evidence for involvement of splicing regulatory proteins

• Published in: RNA biology Vol. 11; no. 11; pp. 1430 - 1446
• Main Authors: Huo, Qing, Kayikci, Melis, Odermatt, Philipp, Meyer, Kathrin, Michels, Olivia, Saxena, Smita, Ule, Jernej, Schümperli, Daniel
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 02.11.2014
• Subjects: Animals; Blotting, Western; Cell Line, Tumor; Cells, Cultured; exon junction microarray; Gene Expression Regulation; Humans; Introns - genetics; laser capture microdissection; major spliceosome; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mice; Mice, Knockout; Mice, Transgenic; minor spliceosome; motoneurons; Motor Neurons - metabolism; Muscular Atrophy, Spinal - genetics; Muscular Atrophy, Spinal - metabolism; Muscular Atrophy, Spinal - pathology; Neuroblastoma - genetics; Neuroblastoma - metabolism; Neuroblastoma - pathology; neurodegerative disease; Research Papers; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA Splicing; RNA Splicing Factors; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Septins - genetics; Septins - metabolism; SMN Complex Proteins - genetics; SMN Complex Proteins - metabolism; snRNP assembly; Spinal Muscular Atrophy; splicing; splicing regulators; NMD, nonsense-mediated mRNA decay; RT, reverse transcription; snRNA, small nuclear ribonucleic acid; Spinal Muscular Atrophy; laser capture microdissection; snRNP assembly; SMN, Survival Motor Neuron; minor spliceosome; real-time (quantitative) PCR; SMA, Spinal Muscular Atrophy; ESE, exonic splicing enhancer; splicing; exon junction microarray; polymerase chain reaction, qPCR; motoneurons; neurodegerative disease; MN, motoneuron; TcRβ, T-cell receptor β chain; hz, heterozygote, LCM; snRNP, small nuclear ribonucleoprotein; major spliceosome; NMJ, neuromuscular junction, PCR; sh, short hairpin; splicing regulators; FCS, fetal calf serum
• ISSN: 1547-6286; 1555-8584
• DOI: 10.1080/15476286.2014.996494

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.