Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes

Abstract

Humans have two nearly identical copies of the survival motor neuron (SMN ) gene, SMN1 and SMN2. Homozygous loss of SMN1 causes spinal muscular atrophy (SMA). SMN2 is unable to prevent the disease due to skipping of exon 7. Using a systematic approach of in vivo selection, we have previously demonstrated that a weak 5′ splice site (ss) serves as the major cause of skipping of SMN2 exon 7. Here we show the inhibitory impact of RNA structure on the weak 5′ ss of exon 7. We call this structure terminal stem–loop 2 (TSL2). Confirming the inhibitory nature of TSL2, point mutations that destabilize TSL2 promote exon 7 inclusion in SMN2, whereas strengthening of TSL2 promotes exon 7 skipping even in SMN1. We also demonstrate that TSL2 negatively affects the recruitment of U1snRNP at the 5′ ss of exon 7. Using enzymatic structure probing, we confirm that the sequence at the junction of exon 7/intron 7 folds into TSL2 and show that mutations in TSL2 cause predicted structural changes in this region. Our findings reveal for the first time the critical role of RNA structure in regulation of alternative splicing of human SMN.

The Silent Sway of Splicing by Synonymous Substitutions

“…Analysis of Library Mutations-To determine the influence of synonymous mutations on exon inclusion, we used the well studied SMN1 mini-gene, spanning exons 6 -8 (13,17,21) in which exon 7 can be included or excluded depending on the splicing signals within the pre-mRNA. Each set of two neighboring codons in exon 7 was mutated to every possible combination of silent mutations within the context of a hexamer, a minimal binding platform for splicing regulatory proteins (Fig.…”

New insights into RNA secondary structure in the alternative splicing of pre-mRNAs

“…For example, the formation of RNA stem-loop structures close to the 5' splice site in exon 7 of SMN2 promotes exon 7 skipping by interfering with its interaction with the snRNP U1. 40 A second proposed mechanism involves a more indirect effect, in which RNA secondary structures cooperate with ADAR to produce new splice sites ( Fig. 1 and Table 2).…”

Repair of pre-mRNA splicing

“…This region also seems to be involved in a secondary structure that affects exon 7 inclusion. 95 Finally, the last exonic nucleotide represents an A instead of the canonical G found in more than 80% of all human exons. 7 As a consequence, the binding affinity of the U1 snRNP is reduced.…”