A direct interaction between the survival motor neuron protein and p53 and its relationship to spinal muscular atrophy.
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Mutations in the SMN1 (survival motor neuron 1) gene cause spinal muscular atrophy (SMA). We now show that SMN protein, the SMN1 gene product, interacts directly with the tumor suppressor protein, p53. Pathogenic missense mutations in SMN reduce both self-association and p53 binding by SMN, and the extent of the reductions correlate with disease severity. The inactive, truncated form of SMN produced by the SMN2 gene in SMA patients fails to bind p53 efficiently. SMN and p53 co-localize in nuclear Cajal bodies, but p53 redistributes to the nucleolus in fibroblasts from SMA patients. These results suggest a functional interaction between SMN and p53, and the potential for apoptosis when this interaction is impaired may explain motor neuron death in SMA. (+info)
An in vivo reporter system for measuring increased inclusion of exon 7 in SMN2 mRNA: potential therapy of SMA.
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Spinal muscular atrophy (SMA) is a degenerative motor neuron disorder resulting from homozygous loss of the SMN1 gene. SMN2, a nearly identical copy gene, is preserved in SMA patients. A single nucleotide difference between SMN1 and SMN2 causes exon 7 skipping in the majority of SMN2 mRNA. Gene therapy through modulation of SMN2 gene transcription in SMA patients may be possible. We constructed a series of SMN mini-genes comprised of SMN exon 6 to exon 8 sequences fused to green fluorescence protein (GFP) or luciferase reporters, to monitor SMN exon 7 splicing. These reporters recapitulated the splicing patterns of the endogenous SMN gene in stable cell lines. The SMN1-luciferase reporter was approximately 3.5-fold more active than SMN2-luciferase and SMN1-GFP intensities were visually distinguishable from SMN2-GFP. We have screened chemical inducers and inhibitors of kinase pathways using stable SMN-reporter lines and found that the phosphatase inhibitor sodium vanadate specifically stimulated exon 7 inclusion within SMN2 mRNAs. This is the first compound identified that can stimulate exon 7 inclusion into transcripts derived from the endogenous SMN2 gene. These results demonstrate that this system can be utilized to identify small molecules that regulate the splicing of SMN exon 7. (+info)
Gemin5, a novel WD repeat protein component of the SMN complex that binds Sm proteins.
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The survival of motor neurons (SMN) protein is the product of the disease gene of spinal muscular atrophy and is found both in the cytoplasm and the nucleus, where it is concentrated in gems. SMN is part of a multi-protein complex that includes Gemin2, Gemin3, and Gemin4. The SMN complex plays an important role in the cytoplasmic assembly of small nuclear ribonucleoproteins (snRNPs) and likely other RNPs in pre-mRNA splicing and in the assembly of transcriptosomes. Here, we report the identification of an additional component of the SMN complex, a novel WD repeat protein termed Gemin5. Gemin5 binds SMN directly and is a component of the SMN complex. Furthermore, Gemin5 interacts with several of the snRNP core proteins including SmB, SmD1, SmD2, SmD3, and SmE, suggesting that it participates in the activities of the SMN complex in snRNP assembly. Immunolocalization studies demonstrate that Gemin5 is found in the cytoplasm and in the nucleus, where it colocalizes with SMN in gems. The presence of 13 WD repeat domains in the amino-terminal half of Gemin5 and a coiled-coil motif near its carboxyl terminus indicate that it may form a large heteromeric complex and engage in multiple interactions. (+info)
Symmetrical dimethylation of arginine residues in spliceosomal Sm protein B/B' and the Sm-like protein LSm4, and their interaction with the SMN protein.
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Arginine residues in RG-rich proteins are frequently dimethylated posttranslationally by protein arginine methyltransferases (PRMTs). The most common methylation pattern is asymmetrical dimethylation, a modification important for protein shuttling and signal transduction. Symmetrically dimethylated arginines (sDMA) have until now been confined to the myelin basic protein MBP and the Sm proteins D1 and D3. We show here by mass spectrometry and protein sequencing that also the human Sm protein B/B' and, for the first time, one of the Sm-like proteins, LSm4, contain sDMA in vivo. The symmetrical dimethylation of B/B', LSm4, D1, and D3 decisively influences their binding to the Tudor domain of the "survival of motor neurons" protein (SMN): inhibition of dimethylation by S-adenosylhomocysteine (SAH) abolished the binding of D1, D3, B/B', and LSm4 to this domain. A synthetic peptide containing nine sDMA-glycine dipeptides, but not asymmetrically modified or nonmodified peptides, specifically inhibited the interaction of D1, D3, B/B', LSm4, and UsnRNPs with SMN-Tudor. Recombinant D1 and a synthetic peptide could be methylated in vitro by both HeLa cytosolic S100 extract and nuclear extract; however, only the cytosolic extract produced symmetrical dimethylarginines. Thus, the Sm-modifying PRMT is cytoplasmic, and symmetrical dimethylation of B/B', D1, and D3 is a prerequisite for the SMN-dependent cytoplasmic core-UsnRNP assembly. Our demonstration of sDMAs in LSm4 suggests additional functions of sDMAs in tri-UsnRNP biogenesis and mRNA decay. Our findings also have interesting implications for the understanding of the aetiology of spinal muscular atrophy (SMA). (+info)
Regulation of murine survival motor neuron (Smn) protein levels by modifying Smn exon 7 splicing.
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Proximal spinal muscular atrophy (SMA) is caused by mutations in the survival motor neuron gene (SMN1). In humans, two nearly identical copies of SMN exist and differ only by a single non-polymorphic C-->T nucleotide transition in exon 7. SMN1 contains a 'C' nucleotide at the +6 position of exon 7 and produces primarily full-length SMN transcripts, whereas SMN2 contains a 'T' nucleotide and produces high levels of a transcript that lacks exon 7 and a low level of full-length SMN transcripts. All SMA patients lack a functional SMN1 gene but retain at least one copy of SMN2, suggesting that the low level of full-length protein produced from SMN2 is sufficient for all cell types except motor neurons. The murine Smn gene is not duplicated or alternatively spliced. It resembles SMN1 in that the critical exon 7 +6 'C' nucleotide is conserved. We have generated Smn minigenes containing either wild-type Smn exon 7 or an altered exon 7 containing the C-->T nucleotide transition to mimic SMN2. When expressed in cultured cells or transgenic mice, the wild-type minigene produced only full-length transcripts whereas the modified minigene alternatively spliced exon 7. Furthermore, Smn exon 7 contains a critical AG-rich exonic splice enhancer sequence (ESE) analogous to the human ESE within SMN exon 7, and subtle mutations within the mESE caused a variation in Smn transcript levels. In summary, we show for the first time that the murine Smn locus can be induced to alternatively splice exon 7. These results demonstrate that SMN protein levels can be varied in the mouse by the introduction of specific mutations at the endogenous Smn locus and thereby lay the foundation for developing animals that closely 'resemble' SMA patients. (+info)
Aclarubicin treatment restores SMN levels to cells derived from type I spinal muscular atrophy patients.
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Proximal spinal muscular atrophy (SMA) is a common motor neuron disorder caused by mutation of the telomeric survival of motor neuron gene SMN1. The centromeric survival of motor neuron SMN2 gene is retained in all SMA patients but does not produce sufficient SMN protein to prevent the development of clinical symptoms. The SMN1 and SMN2 genes differ functionally by a single nucleotide change. This change affects the efficiency with which exon 7 is incorporated into the mRNA transcript. Thus, SMN2 produces less full-length mRNA and protein than SMN1. We have screened a library of compounds in order to identify ones that can alter the splicing pattern of the SMN2 gene. Here, we report that the compound aclarubicin increases the retention of exon 7 into the SMN2 transcript. We show that aclarubicin effectively induces incorporation of exon 7 into SMN2 transcripts from the endogenous gene in type I SMA fibroblasts as well as into transcripts from a SMN2 minigene in the motor neuron cell line NSC34. In type I fibroblasts, treatment resulted in an increase in SMN protein and gems to normal levels. Our results suggest that alteration of splicing pattern represents a new approach to modification of gene expression in disease treatment and demonstrate the feasibility of high throughput screens to detect compounds that affect the splicing pattern of a gene. (+info)
Specific interaction of Smn, the spinal muscular atrophy determining gene product, with hnRNP-R and gry-rbp/hnRNP-Q: a role for Smn in RNA processing in motor axons?
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Spinal muscular atrophy (SMA), the most common hereditary motor neuron disease in children and young adults is caused by mutations in the telomeric survival motor neuron (SMN1) gene. The human genome, in contrast to mouse, contains a second SMN gene (SMN2) which codes for a gene product which is alternatively spliced at the C-terminus, but also gives rise to low levels of full-length SMN protein. The reason why reduced levels of the ubiquitously expressed SMN protein lead to specific motor neuron degeneration without affecting other cell types is still not understood. Using yeast two-hybrid techniques, we identified hnRNP-R and the highly related gry-rbp/hnRNP-Q as novel SMN interaction partners. These proteins have previously been identified in the context of RNA processing, in particular mRNA editing, transport and splicing. hnRNP-R and gry-rbp/hnRNP-Q interact with wild-type Smn but not with truncated or mutant Smn forms identified in SMA. Both proteins are widely expressed and developmentally regulated with expression peaking at E19 in mouse spinal cord. hnRNP-R binds RNA through its RNA recognition motif domains. Interestingly, hnRNP-R is predominantly located in axons of motor neurons and co-localizes with Smn in this cellular compartment. Thus, this finding could provide a key to understand a motor neuron-specific Smn function in SMA. (+info)
Quantitative analyses of SMN1 and SMN2 based on real-time lightCycler PCR: fast and highly reliable carrier testing and prediction of severity of spinal muscular atrophy.
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Spinal muscular atrophy (SMA) is a common autosomal recessive disorder in humans, caused by homozygous absence of the survival motor neuron gene 1 (SMN1). SMN2, a copy gene, influences the severity of SMA and may be used in somatic gene therapy of patients with SMA in the future. We present a new, fast, and highly reliable quantitative test, based on real-time LightCycler PCR that amplifies either SMN1 or SMN2. The SMN1 copies were determined and validated in 329 carriers and controls. The specificity of the test is 100%, whereas the sensitivity is 96.2%. The quantitative analysis of SMN2 copies in 375 patients with type I, type II, or type III SMA showed a significant correlation between SMN2 copy number and type of SMA as well as duration of survival. Thus, 80% of patients with type I SMA carry one or two SMN2 copies, and 82% of patients with type II SMA carry three SMN2 copies, whereas 96% of patients with type III SMA carry three or four SMN2 copies. Among 113 patients with type I SMA, 9 with one SMN2 copy lived <11 mo, 88/94 with two SMN2 copies lived <21 mo, and 8/10 with three SMN2 copies lived 33-66 mo. On the basis of SMN2 copy number, we calculated the posterior probability that a child with homozygous absence of SMN1 will develop type I, type II, or type III SMA. (+info)