Abstract
Spinal muscular atrophy (SMA) is an intractable neurodegenerative disease afflicting 1 in 6–10,000 live births. One of the key functions of the SMN protein is regulation of spliceosome assembly. Reduced levels of the SMN protein that are observed in SMA have been shown to result in aberrant mRNA splicing. SMN-dependent mis-spliced transcripts in motor neurons may cause stresses that are particularly harmful and may serve as potential targets for the treatment of motor neuron disease or as biomarkers in the SMA patient population. We performed deep RNA sequencing using motor neuron-like NSC-34 cells to screen for SMN-dependent mRNA processing changes that occur following acute depletion of SMN. We identified SMN-dependent splicing changes, including an intron retention event that results in the production of a truncated Rit1 transcript. This intron-retained transcript is stable and is mis-spliced in spinal cord from symptomatic SMA mice. Constitutively active Rit1 ameliorated the neurite outgrowth defect in SMN depleted NSC-34 cells, while expression of the truncated protein product of the mis-spliced Rit1 transcript inhibited neurite extension. These results reveal new insights into the biological consequence of SMN-dependent splicing in motor neuron-like cells.
Highlights
Addition of doxycycline (2 μg/ml) for 72 hours induced expression of the survival motor neuron (SMN) shRNA, which resulted in a ~70% reduction of the murine SMN protein as determined by Western blot
Consistent with the results seen in SMN-depleted NIH-3T3 cells, we detected significantly altered U snRNP mRNA levels (p = 0.0013 by one-way ANOVA), with post-hoc Tukey analysis revealing significantly decreased levels of U11 and U4 (p
Our interpretation of the absence of overlap in the two model systems is that while distinct SMN-dependent splicing changes are present among the test systems, the lack of a consistent constellation of errors implies that this cannot be the dominant cause of Spinal muscular atrophy (SMA) pathology, in our model system, the protein product resulting from aberrant Rit1 splicing appears to be a potential explanation for the reduction in neurite length seen after SMN depletion
Summary
MISO was used to analyze intronic sequence retention from NSC-34 transcriptome reads comparing cells with wild type and depleted levels of SMN protein. Primers anchored in the flanking exons show a strong RT-PCR product under all conditions that was unaffected by SMN depletion (Fig 3a), demonstrating that SMN-dependent intron retention events are specific to individual targets rather than a more widespread splicing error.
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