Spinal muscular atrophy (SMA) is a developmental disorder characterized by a progressive loss of motor neurons during early childhood. Based on the frequency of occurrence, SMA is ranked as the second leading genetic cause of infant mortality after cystic fibrosis. Most cases of SMA are associated with the low levels of SMN protein due to deletion or mutation of Survival Motor Neuron 1 (SMN1) gene. A nearly identical copy of the gene, SMN2, fails to compensate for the loss of SMN1 owing to predominant SMN2 exon 7 skipping that produces a truncated protein, SMN?7. A single missense mutation (E134K) in tudor domain of SMN has been also linked to SMA. Among several important functions, SMN has been implicated in biogenesis of small-nuclear ribonucleoproteins (snRNPs), transcription, pre-mRNA splicing, macromolecular trafficking, signal transduction and stress granule formation. SMN contains a distinct nucleic acid binding domain that has been shown to have preference for poly-guanosine (poly rG) RNAs in vitro. We have recently concluded an in vitro selection experiment that revealed diversity of sequence motifs recognized by SMN. These results support a wider role of SMN through direct interactions with a variety of cellular transcripts (RNAs). Here we will perform a systematic analysis of transcriptome-wide interactions of SMN using powerful approaches of UV crosslinking and immunoprecipitation (CLIP) and high throughput sequencing.
In Aim 1, we will perform CLIP experiments to capture transcriptome-wide interactions of SMN in neuronal SH-SY5Y cells. We will optimize UV-crosslinking conditions by modifying various parameters, including use of photoreactive ribonucleosides as in PAR-CLIP (Photoactivatable- Ribonucleoside-Enhanced CLIP). We will employ high throughput sequencing to analyze CLIP tags (crosslinked sequences) associated with SMN. To analyze those sequences that are not amplifiable in CLIP/PAR-CLIP, we will employ iCLIP (individual nucleotide resolution UV-CLIP). To determine the nature of cellular RNAs interacting with SMN, we will map CLIP tags to human genome. Using genomic mapping, we will determine crosslink-induced mutation sites (CIMS) that will help identify motifs responsible for SMN interaction with a single-nucleotide precision.
In aim 2, we will validate the functional significance of novel RNA-SMN interactions revealed by CLIP tags and CIMS data. Severity of SMA is affected by level of SMN (lower the SMN levels higher the severity). Therefore, we will assess the alterations in the transcriptome-wide interactions of SMN at reduced levels of SMN. Findings of this study will reveal signature of SMN-interacting transcripts that are drastically altered at reduced SMN concentrations. To assess that the transcriptome-wide interactions of disease-associated mutant SMN proteins are distinct from the wild type SMN, we will perform CLIP/PAR-CLIP/iCLIP experiments with SMN?7 and E134K. To uncover the possible mechanism by which RNA-SMN interactions affect splicing, stability and trafficking of specific transcripts, we will perform cell-based experiments with reporter assays. Also, we will validate the key findings of our CLIP experiments in motor neurons obtained from control and SMA mice. Our proposal has potential to identify novel SMN functions with significance to a better understanding of molecular mechanism of SMA pathogenesis. )
Deficiency of Survival Motor Neuron (SMN) protein during development causes spinal muscular atrophy (SMA), one of the leading genetic causes of infant mortality. In this proposal, we will perform global analysis of RNA-SMN interactions to identify novel SMN functions with significance to a better understanding of molecular mechanism of SMA pathogenesis.)
|Ottesen, Eric W; Howell, Matthew D; Singh, Natalia N et al. (2016) Severe impairment of male reproductive organ development in a low SMN expressing mouse model of spinal muscular atrophy. Sci Rep 6:20193|
|Seo, Joonbae; Singh, Natalia N; Ottesen, Eric W et al. (2016) Oxidative Stress Triggers Body-Wide Skipping of Multiple Exons of the Spinal Muscular Atrophy Gene. PLoS One 11:e0154390|
|Seo, Joonbae; Singh, Natalia N; Ottesen, Eric W et al. (2016) A novel human-specific splice isoform alters the critical C-terminus of Survival Motor Neuron protein. Sci Rep 6:30778|
|Singh, Natalia N; Lee, Brian M; Singh, Ravindra N (2015) Splicing regulation in spinal muscular atrophy by an RNA structure formed by long-distance interactions. Ann N Y Acad Sci 1341:176-87|
|Singh, Natalia N; Lee, Brian M; DiDonato, Christine J et al. (2015) Mechanistic principles of antisense targets for the treatment of spinal muscular atrophy. Future Med Chem 7:1793-808|
|Seo, Joonbae; Ottesen, Eric W; Singh, Ravindra N (2014) Antisense methods to modulate pre-mRNA splicing. Methods Mol Biol 1126:271-83|
|Howell, Matthew D; Singh, Natalia N; Singh, Ravindra N (2014) Advances in therapeutic development for spinal muscular atrophy. Future Med Chem 6:1081-99|
|Singh, Natalia N; Lawler, Mariah N; Ottesen, Eric W et al. (2013) An intronic structure enabled by a long-distance interaction serves as a novel target for splicing correction in spinal muscular atrophy. Nucleic Acids Res 41:8144-65|
|Sivanesan, Senthilkumar; Howell, Matthew D; Didonato, Christine J et al. (2013) Antisense oligonucleotide mediated therapy of spinal muscular atrophy. Transl Neurosci 4:|
|Seo, Joonbae; Howell, Matthew D; Singh, Natalia N et al. (2013) Spinal muscular atrophy: an update on therapeutic progress. Biochim Biophys Acta 1832:2180-90|
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