A feedback loop regulates splicing of the spinal muscular atrophy-modifying gene, SMN2

Department of Cell Biology and Anatomy, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA.
Human Molecular Genetics (Impact Factor: 6.39). 09/2010; 19(24):4906-17. DOI: 10.1093/hmg/ddq425
Source: PubMed

ABSTRACT Spinal muscular atrophy (SMA) is a neurological disorder characterized by motor neuron degeneration and progressive muscle paralysis. The disease is caused by a reduction in survival of motor neuron (SMN) protein resulting from homozygous deletion of the SMN1 gene. SMN protein is also encoded by SMN2. However, splicing of SMN2 exon 7 is defective, and consequently, the majority of the transcripts produce a truncated, unstable protein. SMN protein itself has a role in splicing. The protein is required for the biogenesis of spliceosomal snRNPs, which are essential components of the splicing reaction. We now show that SMN protein abundance affects the splicing of SMN2 exon 7, revealing a feedback loop inSMN expression. The reduced SMN protein concentration observed in SMA samples and in cells depleted of SMN correlates with a decrease in cellular snRNA levels and a decrease in SMN2 exon 7 splicing. Furthermore, altering the relative abundance or activity of individual snRNPs has distinct effects on exon 7 splicing, demonstrating that core spliceosomal snRNPs influence SMN2 alternative splicing. Our results identify a feedback loop in SMN expression by which low SMN protein levels exacerbate SMN exon 7 skipping, leading to a further reduction in SMN protein. These results imply that a modest increase in SMN protein abundance may cause a disproportionately large increase in SMN expression, a finding that is important for assessing the therapeutic potential of SMA treatments and understanding disease pathogenesis.

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Available from: Dominik Duelli, Sep 27, 2015
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    • "#2) Inefficient inclusion of SMN2 exon 7 has recently been identified as an intrinsic property of motor neurons under normal conditions [25]. Furthermore, when SMN levels are diminished this inclusion becomes even less efficient [22] [25]. This negative feedback loop, where low SMN levels lead to a reduction in snRNP levels, which in turn decreases SMN2 exon 7 incorporation further reducing the amount of available SMN protein, appears to be specific to SMA motor neurons [25]. "
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    • "It is now clearly proven that the SMN complex plays an essential role for assembly of Sm- and Lsm-containing RNPs, i.e. minor and major UsnRNPs, U7 snRNP and Herpesvirus saimiri HSURs (18,19,22–27,86–88). As a result, SMN deficiency was found to alter the repertoire of UsnRNAs in cells and tissues derived from SMA mice, in HeLa cells depleted of SMN by RNAi, as well as in the S. pombe mutant strain carrying a temperature-degron allele of SMN (28–30,32,89,90). Similarly, an implication of the SMN complex in SRP biogenesis was expected to result in SRP assembly defects in SMN-deficient cells. "
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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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    • "Dying mice likely suffer from hypoxia; hypoxic stress induces reactive oxygen species generation, which inactivates the SMN complex (Wan et al. 2008). This complex plays fundamental roles in assembling snRNPs, which are required for splicing (Burghes and Beattie 2009), and low SMN levels result in decreased SMN2 exon 7 splicing through a feedback loop (Jodelka et al. 2010; Ruggiu et al. 2012). Thus, under critical dying conditions, including malnutrition and hypoxia, SMA may progress with gradual , widespread splicing alterations in which the resulting SMN dysfunction and deficiency could be partly involved. "
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    ABSTRACT: Antisense oligonucleotides (ASOs) are versatile molecules that can be designed to specifically alter splicing patterns of target pre-mRNAs. Here we exploit this feature to phenocopy a genetic disease. Spinal muscular atrophy (SMA) is a motor neuron disease caused by loss-of-function mutations in the SMN1 gene. The related SMN2 gene expresses suboptimal levels of functional SMN protein due to alternative splicing that skips exon 7; correcting this defect-e.g., with ASOs-is a promising therapeutic approach. We describe the use of ASOs that exacerbate SMN2 missplicing and phenocopy SMA in a dose-dependent manner when administered to transgenic Smn(-/-) mice. Intracerebroventricular ASO injection in neonatal mice recapitulates SMA-like progressive motor dysfunction, growth impairment, and shortened life span, with α-motor neuron loss and abnormal neuromuscular junctions. These SMA-like phenotypes are prevented by a therapeutic ASO that restores correct SMN2 splicing. We uncovered starvation-induced splicing changes, particularly in SMN2, which likely accelerate disease progression. These results constitute proof of principle that ASOs designed to cause sustained splicing defects can be used to induce pathogenesis and rapidly and accurately model splicing-associated diseases in animals. This approach allows the dissection of pathogenesis mechanisms, including spatial and temporal features of disease onset and progression, as well as testing of candidate therapeutics.
    Genes & development 08/2012; 26(16):1874-84. DOI:10.1101/gad.197418.112 · 10.80 Impact Factor
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