Characterization of a complex regulatory element of Spinal Muscular Atrophy genes

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DESCRIPTION (provided by applicant): Humans have two nearly identical copies of Survival Motor Neuron (SMN) gene, SMN1 and SMN2. Low SMN levels due to deletion and/or mutation of SMN1 lead to spinal muscular atrophy (SMA), a major genetic cause of infant mortality. SMN2 fails to compensate for the loss of SMN1 due to a C to T mutation at the 6th position (C6U in transcript) in exon 7. C6U weakens the 32-splice site and triggers SMN2 exon 7 skipping, resulting in synthesis of a truncated protein (SMN¿7), which is unstable. It is known that strategies aimed at correction of SMN2 exon 7 splicing hold the promise for a cure. This proposal emanates from our recent discovery of a unique RNA structure formed by a long-distance interaction (LDI) as a regulator of SMN2 exon 7 splicing (Singh et al., Nucleic Acids Res., 2013, doi:10.1093/nar/gkt609). We call this structure internal stem through LDI-1 (ISTL1). Employing the SHAPE (Selective 22-Hydroxyl Acylation analyzed by Primer Extension) method, we confirmed the formation and functional significance of ISTL1. We showed that an antisense oligonucleotide (ASO)-mediated sequestration of the 32 strand of ISTL1 fully corrects SMN2 exon 7 splicing and restores high levels of SMN and Gemin2, an SMN-interacting protein, in SMA patient cells. Our results also revealed that the 32 strand of ISTL1 is located within a large inhibitory region that we termed intronic splicing silencer N2 (ISS-N2). To continue with our lead, here we propose to characterize additional (novel) intronic cis-elements and their cognate transacting factors in regulation of SMN2 exon 7 splicing. A part of the proposal is aimed at validating the therapeutic efficacy of ISS-N2- targeting ASOs. In Aim 1, we will use overlapping deletions, ASO-based strategies and SHAPE analyses to determine the significance of novel cis-elements within SMN2 intron 7. We will validate our findings in different cell types including motor neuron-like NSC34 cells. We will examine the effect of a larger structural context on the accessibility of the splice sites of SMN2 exon 7. We will determine whether SMN2-specific mutations lead to a structural difference between SMN1 and SMN2 pre-mRNAs, particularly at the splice sites of SMN exon 7. In addition, we will evaluate whether critical cis-elements within intron 6 affect the structural context of intron 7 and potentially lead to the remodeling of the 52 slice site of exon 7. In Aim 2, we will employ over-expression and siRNA-based strategies to identify splicing factors that assist ISTL1 formation and/or use ISTL1 as a site for self-recruitment. We will use a biotinylated oligonucleotide as a trapping device to capture novel RNA-protein complexes that are deposited on SMN2 intron 7 and are critical for the inhibitory effect of LDI. We have previously shown that TIA1 stimulates SMN2 exon 7 splicing by binding to intron 7. We also demonstrated critical role of the Q-rich domain of TIA1 in regulation of SMN2 exon 7 splicing. Recently, a point mutation within Q-rich domain of TIA1 has been shown to cause Welander distal myopathy as well as promote SMN2 exon 7 skipping. Based on these findings and our preliminary results, we will examine the role of another Q-rich domain containing splicing regulator, SFPQ, in SMN2 exon 7 splicing. We will employ UV-crosslinking, footprinting, in vitro binding and SHAPE-based approaches to characterize RNA-protein interactions. In Aim 3, we will perform in vivo studies in a mild as well as in a severe mouse model of SMA to determine the therapeutic efficacy of an ISS-N2 targeting lead phosphorodiamidate morpholino oligomer (PMO). We will design our experimental plan based on several successful in vivo studies reported recently and will employ rigorous criteria of sample-size estimation, randomization and blinding. A successful outcome will lead to the development of a novel ASO-based therapy for SMA.