Structural Context of a Critical Exon of Spinal Muscular Atrophy Gene

Abstract

Humans contain two nearly identical copies of Survival Motor Neuron genes, SMN1 and SMN2. Deletion or mutation of SMN1 causes spinal muscular atrophy (SMA), one of the leading genetic diseases associated with infant mortality. SMN2 is unable to compensate for the loss of SMN1 due to predominant exon 7 skipping, leading to the production of a truncated protein. Antisense oligonucleotide and small molecule-based strategies aimed at the restoration of SMN2 exon 7 inclusion are approved therapies of SMA. Many cis-elements and transacting factors have been implicated in regulation of SMN exon 7 splicing. Also, several structural elements, including those formed by a long-distance interaction, have been implicated in the modulation of SMN exon 7 splicing. Several of these structures have been confirmed by enzymatic and chemical structure-probing methods. Additional structures formed by inter-intronic interactions have been predicted by computational algorithms. SMN genes generate a vast repertoire of circular RNAs through inter-intronic secondary structures formed by inverted Alu repeats present in large number in SMN genes. Here, we review the structural context of the exonic and intronic cis-elements that promote or prevent exon 7 recognition. We discuss how structural rearrangements triggered by single nucleotide substitutions could bring drastic changes in SMN2 exon 7 splicing. We also propose potential mechanisms by which inter-intronic structures might impact the splicing outcomes.

Keywords: ISS-N1; RNA structure; SMA; SMN; small molecule; spinal muscular atrophy; splicing; survival motor neuron.

FIGURE 1

Regulation of SMN exon 7 splicing. Diagrammatic representation of intronic and exonic cis-elements as well as trans-acting factors that modulate SMN exon 7 splicing. Upper-case letters signify exonic sequences, small-case letters, intronic sequences. Exons and introns are also shown as colored boxes and lines, respectively. Numbering of nucleotides, neutral and positive, starts from the first exonic and intronic position, respectively. The 5 and 3′ splice sites (5′ss and 3′ss) are indicated by the arrows. Exinct, conserved tract and the 3′-Cluster are cis-elements revealed by in vivo selection as described in (Singh et al., 2004b). Cr1 and Cr2 represent cryptic 5′ splice sites as described in (Singh et al., 2017a). Negative and positive regulators of exon 7 splicing are indicated by (−) and (+), respectively. Abbreviations: Exinct, extended inhibitory context, GCRS, GC-rich region; LDI, long-distance interaction; URC, Uridine-rich clusters.

FIGURE 2

Local structure of SMN exon 7 and adjacent upstream/downstream intronic sequences. Existence of TSL2 and its effect on exon 7 splicing was also confirmed by mutational analysis. Intron 6 and intron 7 sequences are shown in small-case green and blue letters, respectively. Exon 7 sequence is shown in upper-case black letters. Numbering of nucleotides, neutral, positive and negative, starts from the first position in exon 7, first position of intron 7 and the last position in intron 6, respectively. The splice sites of exon 7 are indicated by the arrows. IS1 structure is boxed. Abbreviation: IL, internal loop; IS, internal stem; TSL, terminal stem loop.

FIGURE 3

Secondary structure of SMN intron 7. The structure is based on combined probing by enzymatic and chemical methods. An exon 7/intron 7 junction as well as the 5′ss are indicated. Exon 8 is represented by a green box. Numbering of nucleotides, neutral and. positive, starts from the first position of exon 7 and the first position of intron 7, respectively. Binding sites of hnRNP A1/A2 and TIA1 are highlighted in pink and green, respectively. Element 2 sequence is highlighted in light blue. ISTL, internal stem formed by a long-distance interaction; TSL, terminal stem-loop; ISS-N2, intronic splicing silencer 2.

FIGURE 4

Predicted inter-intronic structures. (A). Secondary structure formed between elements 1 and 2 located within introns 6 and 7, respectively. The structure sequesters both TIA1 binding sites. Numbering of nucleotides, negative, neutral and negative, starts from the last position of intron 6, first position of exon 7 and the first position of intron 7, respectively. Element 1 is highlighted in purple; branch point sequence, in yellow, with “A” indicated in red. Other markings and abbreviations are same as shown in Figure 3. (B). An alternative secondary structure of element 2. The structural context changes the positioning of the TIA1 binding sites. Markings and abbreviation are the same as in panel A.

FIGURE 5

In silico ScanFold results for the pre-mRNA of SMN2. (A). At the top, an IGV representation of the whole SMN2 pre-mRNA transcript with 6 data tracks: a base pair (arc diagram) track, a track showing ScanFold extracted structures where base pairs with z-score < -2, -1 and 0 are indicated in blue, green and yellow, respectively; a track of transcripts (with introns as lines and exons as boxes), an ensemble diversity (ED) track, a minimum free energy (MFE) track, and a ΔG z-score track. Below the whole transcript are two zoomed in regions. Highlighted by the blue box is a region of SMN2 with the lowest z-score and both the lowest and highest MFE regions in the transcript. Highlighted by the red box is Exon 7, which contains one of the two extracted structures present in an exonic region. (B). The ScanFold informed 2D model of the blue highlighted region from panel A is shown with the per nucleotide (NT) z-score overlaid on the model. (C). The ScanFold informed 2D model of the red highlighted region from panel A is shown with the per nucleotide (NT) z-score overlaid on the model. Here, the boundaries of intron splice sites and the start and stop site of exon 7 are labelled.