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Review
. 2010 Jul-Aug;7(4):430-40.
doi: 10.4161/rna.7.4.12206. Epub 2010 Jul 1.

Repair of pre-mRNA splicing: prospects for a therapy for spinal muscular atrophy

Rachel Nlend Nlend  1 , Kathrin Meyer, Daniel Schümperli
Affiliations
Review

Repair of pre-mRNA splicing: prospects for a therapy for spinal muscular atrophy

Rachel Nlend Nlend et al. RNA Biol. 2010 Jul-Aug.

Abstract

Recent analyses of complete genomes have revealed that alternative splicing became more prevalent and important during eukaryotic evolution. Alternative splicing augments the protein repertoire--particularly that of the human genome--and plays an important role in the development and function of differentiated cell types. However, splicing is also extremely vulnerable, and defects in the proper recognition of splicing signals can give rise to a variety of diseases. In this review, we discuss splicing correction therapies, by using the inherited disease Spinal Muscular Atrophy (SMA) as an example. This lethal early childhood disorder is caused by deletions or other severe mutations of SMN1, a gene coding for the essential survival of motoneurons protein. A second gene copy present in humans and few non-human primates, SMN2, can only partly compensate for the defect because of a single nucleotide change in exon 7 that causes this exon to be skipped in the majority of mRNAs. Thus SMN2 is a prime therapeutic target for SMA. In recent years, several strategies based on small molecule drugs, antisense oligonucleotides or in vivo expressed RNAs have been developed that allow a correction of SMN2 splicing. For some of these, a therapeutic benefit has been demonstrated in mouse models for SMA. This means that clinical trials of such splicing therapies for SMA may become possible in the near future.

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Figures

Figure 1
Figure 1
Cis acting sequence elements and trans-acting factors determining exon definition. (A) Conserved sequences flanking metazoan and yeast exons. The exon is symbolized by a yellow rectangle. The flanking introns are symbolized by lines in which the branch point (BP) sequence and polypyrimidine tract (PP tract) are highlighted as light blue boxes. The conserved nucleotides of the BP, PP tract, 3′ splice site (3′ SS) and 5′ splice site (5′ SS) are shown below with numbers indicating the percent prevalence of the most frequent nucleotides at each position. Shown in red are the branch point adenosine as well as the virtually invariant last two and first two nucleotides of the introns. (B) Role of splicing activators and repressors in splicing modulation. SR proteins bind to exonic splicing enhancers (ESE; dark blue) via their RNA recognition motifs and favour the recruitment of the splicing machinery (+) at the 5′ and 3′ SS, mainly by stabilizing the interaction between snRNPs and the pre-mRNA. SR proteins also act by direct protein-protein interactions with U1 snRNPs at the 5′ SS, and with the U2 snRNP and U2AF co-factors at the 3′ SS. In contrast, hnRNP proteins most frequently bind to intronic splicing silencers (ISS; red) and are involved in repression (−) of splicing, by exerting negative effects on U snRNPs or SR proteins. These different interactions are represented by arrows.
Figure 2
Figure 2
Splicing architecture of exon 7 of the human SMN1 and SMN2 genes. The diagram represents exon 7 (yellow box) and its flanking intronic regions (lines). Elements inhibiting exon 7 inclusion are shown in red, whereas the positive elements are represented in dark blue. The suboptimal branch point (BP) and polypyrimidine tract (PP tract) are indicated in light blue. SF2/ASF and Tra2/β1 bind to the exonic splicing enhancers SE1 and SE2, respectively. The recognition of SE1 by SF2/ASF is prevented in SMN2, due to the C → U transition. This sequence alteration also creates a hnRNP A1-dependent splicing silencer (see main text for refs.).
Figure 3
Figure 3
Schematic representation of the different AON-based splicing correction strategies for SMA. Red and blue arrows indicate negative and positive splicing effects, respectively. Small blue dots signify unspecified splicing factors necessary for exon definition and splicing. (A) In a concept of 3′ SS competition where exon 8 prevails over exon 7, an AON masking the 3′ SS of exon 8 will partly shift splicing factor recruitment to exon 7. (B) Masking any of the flanking ISS by an AON can stimulate exon 7 inclusion in the mRNA. (C) The bifunctional strategy allows to tether binding sequences for different SR proteins to either ISS element 1 or the altered SE1 sequence and thereby to enhance the recruitment of the splicing machinery to exon 7. As the mutated SE1 element is also a splicing silencer, both approaches have dual effects by masking a negatively acting element and by recruiting positively acting SR proteins. An AON targeting the exon 8 3′ SS that bears a tail with a binding sequence for hnRNP A1 will also more efficiently shift splicing factor recruitment to exon 7 than the corresponding tail-less AON (see main text for refs.).
Figure 4
Figure 4
Scheme depicting splicing correction strategies for SMA based on in vivo expressed RNAs (A) SnRNA-based strategies. Bifunctional U7 Sm OPT derivatives have been designed to tether SR proteins to various positions in exon 7. Alternatively, U7 Sm OPT derivatives can mask the BP and 3′ SS of exon 8, either with or without tethering hnRNP A1. Shown with dark grey vertical arrows are snRNA-based strategies that were either not successful (U2 snRNA fully complementary to BP upstream of exon 7), inhibitory (U7 Sm OPT targeting ISS-N1) or toxic to cells (U1 snRNA fully complementary to the exon 7 5′ SS). Inset: Basic structure of a U7 Sm OPT derivative. The important elements are (from 5′ to 3′): the antisense sequence, the Sm OPT site capable of assembling with a heptameric Sm core of the standard Sm proteins (nucleotide changes respective to wild-type U7 snRNA are shown in red), and a 3′-terminal hairpin which stabilises the RNA. Splicing enhancer or silencer sequences can be added at the 5′ end to generate bifunctional U7 snRNAs. Note that transcription from a U snRNA promoter is important to allow efficient 3′ end formation at the U snRNA 3′ box and assembly into a snRNP particle. Moreover, mammalian U7 snRNAs start with an adenosine residue. (B) Trans-splicing strategy. A SMN-specific trans-splicing RNA (tsRNA) will bind to the BP/3′ SS region upstream of SMN2 exon 7 (for simplicity only the BP is shown) and will contain a strong BP/3′ SS leading into a SMN1-specific version of exon 7 (shown in green) and ending in a poly(A) tail downstream of the stop codon. After splicing, this SMN1-specific exon 7 will be fused to the body of the endogenous SMN2 mRNAs containing exons 1–6. Black knobs indicate the cap structures at the 5′ ends of the RNAs involved (see main text for refs.).
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