Abnormal Golgi morphology and decreased COPI function in cells with low levels of SMN

We report here the finding of abnormal Golgi apparatus morphology in motor neuron like cells depleted of SMN as well as Golgi apparatus morphology in SMA patient fibroblasts. Rescue experiments demonstrate that this abnormality is dependent on SMN, but can also be rescued by expression of the COPI coatomer subunit alpha-COP. A motor neuron-like cell line containing an inducible alpha-COP shRNA was created to generate a parallel system to study knockdown of SMN or alpha-COP. Multiple assays of COPI-dependent intracellular trafficking in cells depleted of SMN demonstrate that alpha-COP function is suboptimal, including failed sequestration of plasma membrane proteins, altered binding of mRNA, and defective targeting and transport of Golgi-resident proteins

Interaction between alpha-COP and SMN ameliorates disease phenotype in a mouse model of spinal muscular atrophy

We report that expression of the α-COP protein rescues disease phenotype in a severe mouse model of Spinal Muscular Atrophy (SMA).. Lentiviral particles expressing α-COP were injected directly into the testes of genetically pure mouse strain of interest resulting in infection of the spermatagonial stem cells. α-COP was stably expressed in brain, skeletal muscle, and spinal cord without altering SMN protein levels. SMA mice transgenic for α-COP live significantly longer than their non-transgenic littermates, and showed increased body mass and normal muscle morphology at postnatal day 15. We previously reported that binding between SMN and α-COP is required for restoration of neurite outgrowth in cells lacking SMN, and we report similar finding here. Lentiviral-mediated transgenic expression of SMN where the dilysine domain in exon 2b was mutated was not able to rescue the SMA phenotype despite robust expression of the mutant SMN protein in brain, muscle and spinal cord. These results demonstrate that α-COP is a validated modifier of SMA disease phenotype in a mammalian, vertebrate model and is a potential target for development of future SMN-independent therapeutic interventions

Altered mRNA Splicing in SMN-Depleted Motor Neuron-Like Cells.

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

SMN-dependent intron skipping events ranked by percent inclusion.

<p>SMN-dependent intron skipping events ranked by percent inclusion.</p

Alternatively spliced Rit1 decreases neurite length.

<p>A) Schematic of Rit1 protein domains. Protein sequence shown of minimal consensus G1-G5 domains of Ras family members on top followed by the mouse Rit1 specific sequence and corresponding amino acid position. SwI: Switch I, SwII: Switch II domains, FL: Full length, TM: truncation mutant. B) Representative levels of SMN for NSC34-4#56 cells used in panels D and E upon dox-induced knockdown. C) Western blot expression of FLAG tagged dominant negative Rit S35N, constitutively active Rit Q79L, full length Rit (FL), and alternatively spliced Rit truncation mutant (TM). D) Neurite extension assay. NSC34-4#56 cells were dox treated for 3 days followed by transfection of FLAG-Rit /GFP for 3 days. Cells were fixed and tubulin stained to allow neurite length measurement in GFP positive cells. Data are representative of three independent experiments, p<0.001 as determined by one-way ANOVA and post-hoc Bonferroni test. **: p<0.01. E) Immunofluorescent images of representative cells. Red: Tubulin, Green: GFP, Blue: DAPI nucleus. F) Western blot from NSC-34 cells following differentiation and 72 hours treatment with doxycycline. Transfection with Rit-79 but not Rit-FL increased levels of phospho-p38 and SMN in doxycycline-treated cultures.</p

SMN depletion results in alternative intron usage.

<p>A) RT-PCR demonstrated increased intron retention events in doxycycline treated NSC-34-4#56, but not NSC-34-4. The histograms show the posterior distributions over PSI estimated by MISO in control or SMN-depleted samples conditions. The red lines depict the posterior mean and the dotted grey lines indicate 95% confidence intervals (also shown in parenthesis). B) Quantification of RT-PCR band intensity from 3 biological replicates shows increased intron retention after SMN depletion, but not after doxycycline treatment of NSC-34-4. *–<i>p</i><0.01. One-way ANOVA followed by post-hoc t-test.</p

SMN depletion leads to defective U-snRNP biogenesis.

<p>A) Doxycycline treatment (2μg/ml) for 72 hrs reduced SMN protein levels by ~70% in NSC-34-4#56, which contain both the RTta and the SMN shRNA but not NSC-34 cell clone 4 which expresses only the RTta. B) Quantification of three separate western blot of SMN protein level after doxycycline treatment in NSC-34-4#56. C) Quantitative RT-PCR shows mRNA levels of UsnRNPs after doxycycline-induced SMN depletion compared to untreated cells (dashed line). All samples were compared to levels of the 18S subunit. *–<i>p</i><0.01. One-way ANOVA followed by post-hoc t-test.</p

SMN-dependent intron retention events ranked by percent inclusion.

<p>SMN-dependent intron retention events ranked by percent inclusion.</p

Aberrant splicing is rescued by reintroduction of human SMN.

<p>A) NSC-34-4#56 cells were transfected with HA-tagged human SMN. Western blotting with antibodies against SMN (MANSMA1) showed that doxycycline reduced the levels of mouse SMN but the HA-hSMN was unaffected and brought levels of SMN protein within normal range after 72 hours in doxycycline. B) RT-PCR on SMN-depleted and HA-hSMN expressing cultures shows rescue of three major splicing change categories. C-D) Quantitative real-time PCR (qRT-PCR) demonstrated that the aberrant splice products from exon skipping/retention (6C) or intron retention (6D) were only present after doxycycline-induced SMN depletion, and normalized with expression of human SMN. Schematic shows the location of primers for qRT-PCR quantification of alternative splice products.</p

RNA-seq workflow and quality control.

<p>RNA-seq workflow and quality control.</p