The Survival of Motor Neurons Protein Determines the Capacity for snRNP Assembly: Biochemical Deficiency in Spinal Muscular Atrophy

Howard Hughes Medical Institute, Department of Biochemistry & Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 08/2005; 25(13):5543-51. DOI: 10.1128/MCB.25.13.5543-5551.2005
Source: PubMed


Reduction of the survival of motor neurons (SMN) protein levels causes the motor neuron degenerative disease spinal muscular atrophy, the severity of which correlates with the extent of reduction in SMN. SMN, together with Gemins 2 to 7, forms a complex that functions in the assembly of small nuclear ribonucleoprotein particles (snRNPs). Complete depletion of the SMN complex from cell extracts abolishes snRNP assembly, the formation of heptameric Sm cores on snRNAs. However, what effect, if any, reduction of SMN protein levels, as occurs in spinal muscular atrophy patients, has on the capacity of cells to produce snRNPs is not known. To address this, we developed a sensitive and quantitative assay for snRNP assembly, the formation of high-salt- and heparin-resistant stable Sm cores, that is strictly dependent on the SMN complex. We show that the extent of Sm core assembly is directly proportional to the amount of SMN protein in cell extracts. Consistent with this, pulse-labeling experiments demonstrate a significant reduction in the rate of snRNP biogenesis in low-SMN cells. Furthermore, extracts of cells from spinal muscular atrophy patients have a lower capacity for snRNP assembly that corresponds directly to the reduced amount of SMN. Thus, SMN determines the capacity for snRNP biogenesis, and our findings provide evidence for a measurable deficiency in a biochemical activity in cells from patients with spinal muscular atrophy.

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    • "Knockdown of snRNP assembly in zebrafish results in degeneration of motor neuron axons [23]. snRNP assembly is defective in SMN-deficient SMA cells [24]. However, this function of SMN in U-type snRNP assembly is required by all cells. "
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    ABSTRACT: Proximal spinal muscular atrophy (SMA) is an early onset, autosomal recessive motor neuron disease caused by loss of or mutation in SMN1 (survival motor neuron 1). Despite understanding the genetic basis underlying this disease, it is still not known why motor neurons (MNs) are selectively affected by the loss of the ubiquitously expressed SMN protein. Using a mouse embryonic stem cell (mESC) model for severe SMA, the RNA transcript profiles (transcriptomes) between control and severe SMA (SMN2+/+;mSmn-/-) mESC-derived MNs were compared in this study using massively parallel RNA sequencing (RNA-Seq). The MN differentiation efficiencies between control and severe SMA mESCs were similar. RNA-Seq analysis identified 3,094 upregulated and 6,964 downregulated transcripts in SMA mESC-derived MNs when compared against control cells. Pathway and network analysis of the differentially expressed RNA transcripts showed that pluripotency and cell proliferation transcripts were significantly increased in SMA MNs while transcripts related to neuronal development and activity were reduced. The differential expression of selected transcripts such as Crabp1, Crabp2 and Nkx2.2 was validated in a second mESC model for SMA as well as in the spinal cords of low copy SMN2 severe SMA mice. Furthermore, the levels of these selected transcripts were restored in high copy SMN2 rescue mouse spinal cords when compared against low copy SMN2 severe SMA mice. These findings suggest that SMN deficiency affects processes critical for normal development and maintenance of MNs.
    PLoS ONE 09/2014; 9(9):e106818. DOI:10.1371/journal.pone.0106818 · 3.23 Impact Factor
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    • "Defective oligomerization of the SMN protein has been causally linked to some cases of spinal muscular atrophy (Lorson et al., 1998; Pellizzoni et al., 1999). In this case, defects in SMN oligomerization cause ineffective assembly of the small nuclear RNPs and disruption of pre-mRNA splicing (Shpargel and Matera, 2005; Wan et al., 2005). Our analysis of Gle1 self-association extends this paradigm for perturbations of RNP effector oligomerization and highlights altered mRNP remodeling as a molecular disease mechanism. "
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    ABSTRACT: The conserved multifunctional protein Gle1 regulates gene expression at multiple steps: nuclear mRNA export, translation initiation, and translation termination. A GLE1 mutation (FinMajor) is causally linked to human lethal congenital contracture syndrome-1 (LCCS1); however, the resulting perturbations on Gle1 molecular function were unknown. FinMajor results in a proline-phenylalanine-glutamine peptide insertion within the uncharacterized Gle1 coiled-coil domain. Here, we find that Gle1 self-associates both in vitro and in living cells via the coiled-coil domain. Electron microscopy reveals that high-molecular-mass Gle1 oligomers form ?26 nm diameter disk-shaped particles. With the Gle1-FinMajor protein, these particles are malformed. Moreover, functional assays document a specific requirement for proper Gle1 oligomerization during mRNA export, but not for Gle1's roles in translation. These results identify a mechanistic step in Gle1's mRNA export function at nuclear pore complexes and directly implicate altered export in LCCS1 disease pathology.
    Cell 10/2013; 155(3):582-93. DOI:10.1016/j.cell.2013.09.023 · 32.24 Impact Factor
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    • "Although reduced levels of SMN cause SMA, it is still unclear why a defect in a ubiquitously expressed protein causes a selective death of the alpha-motor neurons. A defect in the assembly of UsnRNPs is proposed to cause disease onset leading to abnormal splicing of specific mRNAs and disease progression (28–32,87,96,97). Due to the reduction in the levels of minor UsnRNAs observed in several tissues of SMA mice and to the defect in the assembly of the minor tri-snRNP observed in SMA-derived lymphocytes, one can reasonably think that minor splicing pathway could be specifically affected in SMA, and this may concern gene(s) of high importance for motor neurons activity (28,29,31,89). "
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    ABSTRACT: Spinal muscular atrophy is a severe motor neuron disease caused by reduced levels of the ubiquitous Survival of MotoNeurons (SMN) protein. SMN is part of a complex that is essential for spliceosomal UsnRNP biogenesis. Signal recognition particle (SRP) is a ribonucleoprotein particle crucial for co-translational targeting of secretory and membrane proteins to the endoplasmic reticulum. SRP biogenesis is a nucleo-cytoplasmic multistep process in which the protein components, except SRP54, assemble with 7S RNA in the nucleolus. Then, SRP54 is incorporated after export of the pre-particle into the cytoplasm. The assembly factors necessary for SRP biogenesis remain to be identified. Here, we show that 7S RNA binds to purified SMN complexes in vitro and that SMN complexes associate with SRP in cellular extracts. We identified the RNA determinants required. Moreover, we report a specific reduction of 7S RNA levels in the spinal cord of SMN-deficient mice, and in a Schizosaccharomyces pombe strain carrying a temperature-degron allele of SMN. Additionally, microinjected antibodies directed against SMN or Gemin2 interfere with the association of SRP54 with 7S RNA in Xenopus laevis oocytes. Our data show that reduced levels of the SMN protein lead to defect in SRP steady-state level and describe the SMN complex as the first identified cellular factor required for SRP biogenesis.
    Nucleic Acids Research 12/2012; 41(2). DOI:10.1093/nar/gks1224 · 9.11 Impact Factor
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