406 An in vitro splicing assay reveals the pathogenicity of intronic variants in ABCC6, the gene at fault in pseudoxanthoma elasticum

Spinal Muscular Atrophy (SMA) is a devastating neurodegenerative disease caused by reduced amounts of the ubiquitously expressed Survival of Motor Neuron (SMN) protein. In agreement with its crucial role in the biogenesis of spliceosomal snRNPs, SMN-deficiency is correlated to numerous splicing alterations in patient cells and various tissues of SMA mouse models. Among the snRNPs whose assembly is impacted by SMN-deficiency, those involved in the minor spliceosome are particularly affected. Importantly, splicing of several, but not all U12-dependent introns has been shown to be affected in different SMA models. Here, we have investigated the molecular determinants of this differential splicing in spinal cords from SMA mice. We show that the branchpoint sequence (BPS) is a key element controlling splicing efficiency of minor introns. Unexpectedly, splicing of several minor introns with suboptimal BPS is not affected in SMA mice. Using in vitro splicing experiments and oligonucleotides targeting minor or major snRNAs, we show for the first time that splicing of these introns involves both the minor and major machineries. Our results strongly suggest that splicing of a subset of minor introns is not affected in SMA mice because components of the major spliceosome compensate for the loss of minor splicing activity.

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In vitro splicing pathways of pre-mRNAs containing multiple intervening sequences?

Molecular and Cellular Biology ◽

10.1128/mcb.7.10.3428-3437.1987 ◽

1987 ◽

Vol 7 (10) ◽

pp. 3428-3437

Author(s):

K M Lang ◽

R A Spritz

Keyword(s):

Temporal Order ◽

Exon Skipping ◽

Beta Globin ◽

Splice Site Selection ◽

Interleukin 3 ◽

Intervening Sequences ◽

In Vitro Splicing ◽

Lag Times ◽

Alpha Lactalbumin

We analyzed the in vitro splicing pathways of three multi-intervening-sequence (IVS) pre-mRNAs: human beta-globin, which contains two IVSs (K. M. Lang, V. L. van Santen, and R. A. Spritz, EMBO J. 4:1991-1996, 1985); rat alpha-lactalbumin, which contains three IVSs; and murine interleukin-3, which contains four IVSs. We found that there are highly preferred pathways of IVS removal from these multi-IVS pre-mRNAs in vitro. The three IVSs of rat alpha-lactalbumin pre-mRNA were excised sequentially from 5' to 3'; in most molecules, IVS1 was removed first, followed by IVS2 and finally by IVS3. The splicing pathway of interleukin-3 pre-mRNA in vitro was more complex. The four IVSs were excised in a highly preferred temporal order, but the order was not strictly sequential or directional. In most molecules, IVS1 and IVS4 were removed first, either simultaneously or in rapid succession. Subsequently, IVS2 was excised, followed by IVS3. The observed splicing pathways apparently resulted from differences in lag times and maximum excision rates of the different IVSs. We detected no exon skipping during splicing of these transcripts in vitro. These observations have implication for proposed models of splice site selection.

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