PMD patient mutations reveal a long-distance intronic interaction that regulates PLP1/DM20 alternative splicing

Abstract

Alternative splicing of the proteolipid protein 1 gene (PLP1) produces two forms, PLP1 and DM20, due to alternative use of 5' splice sites with the same acceptor site in intron 3. The PLP1 form predominates in central nervous system RNA. Mutations that reduce the ratio of PLP1 to DM20, whether mutant or normal protein is formed, result in the X-linked leukodystrophy Pelizaeus-Merzbacher disease (PMD). We investigated the ability of sequences throughout PLP1 intron 3 to regulate alternative splicing using a splicing minigene construct transfected into the oligodendrocyte cell line, Oli-neu. Our data reveal that the alternative splice of PLP1 is regulated by a long-distance interaction between two highly conserved elements that are separated by 581 bases within the 1071-base intron 3. Further, our data suggest that a base-pairing secondary structure forms between these two elements, and we demonstrate that mutations of either element designed to destabilize the secondary structure decreased the PLP1/DM20 ratio, while swap mutations designed to restore the structure brought the PLP1/DM20 ratio to near normal levels. Sequence analysis of intron 3 in families with clinical symptoms of PMD who did not have coding-region mutations revealed mutations that segregated with disease in three families. We showed that these patient mutations, which potentially destabilize the secondary structure, also reduced the PLP1/DM20 ratio. This is the first report of patient mutations causing disease by disruption of a long-distance intronic interaction controlling alternative splicing. This finding has important implications for molecular diagnostics of PMD.

Mechanism of Splicing Regulation of Spinal Muscular Atrophy Genes

“…In addition, the detailed characterization of ISS-N1 led to the discovery of a unique RNA structure formed by long-distance intra-intronic interactions that contributes to exon 7 skipping. Interestingly, abrogation of a similar structure within intron 3 of the proteolipid protein 1 (PLP1) gene has been recently suggested to cause X-linked Pelizaeus–Merzbacher disease or PMD [128]. Growing evidence suggests that splicing of various exons is differentially regulated under the normal and stress-associated conditions.…”

Conserved long-range base pairings are associated with pre-mRNA processing of human genes

“…from [23] ( Figure S2). The structure responsible for splicing of the intron between exons 9 and 10 in SF1 [33] was in group III (-25.1 kcal/mol); the structure associated with exon 46-52 skipping in DST [34] was in group II (−24.3 kcal/mol); a long-distance intronic interaction that regulates PLP1/DM20 splicing [37] was in group I (−15.8 kcal/mol); the structure that was suggested to regulate mutually exclusive exon choice in DNM1 [38] was in group III (−25.9 kcal/mol). The RNA bridge in ENAH [39] also belongs to group I (−19.4 kcal/mol) and spreads over 1,700 nts, indicating that less stable RNA structures are not less functional or less interesting than the others.…”

Interpretation of mRNA splicing mutations in genetic disease: review of the literature and guidelines for information-theoretical analysis

“…Similarly, variants that strengthen natural splice sites [188][189][190] are also likely to be neutral, though these variants can increase retention of exons that are otherwise frequently alternatively spliced 191,192 . However, binding site variants with minimal splicing information changes may still alter mRNA processing by disrupting mRNA secondary structure 193 .…”