Mutations in U4atac snRNA, a component of the minor spliceosome, in the developmental disorder MOPD I

Abstract

Small nuclear RNAs (snRNAs) are essential factors in messenger RNA splicing. By means of homozygosity mapping and deep sequencing, we show that a gene encoding U4atac snRNA, a component of the minor U12-dependent spliceosome, is mutated in individuals with microcephalic osteodysplastic primordial dwarfism type I (MOPD I), a severe developmental disorder characterized by extreme intrauterine growth retardation and multiple organ abnormalities. Functional assays showed that mutations (30G>A, 51G>A, 55G>A, and 111G>A) associated with MOPD I cause defective U12-dependent splicing. Endogenous U12-dependent but not U2-dependent introns were found to be poorly spliced in MOPD I patient fibroblast cells. The introduction of wild-type U4atac snRNA into MOPD I cells enhanced U12-dependent splicing. These results illustrate the critical role of minor intron splicing in human development.

Fig. 1

Identification of RNU4ATAC mutations in MOPD I patients. (A) High throughput sequencing of ∼1.8 Mb mapped region. Top: Overview of enriched sequence reads from a single MOPD I patient, mapped to the target region showing chromosome 2 coordinates and schematic of genes. Bottom: Paired end reads mapped to targeted region, visualized using IGV browser (Broad Institute), showing the depth of sequencing coverage (gray peaks) at tiled enrichment probes (purple rectangles, bottom); see Supplementary Table 1. Very few sequence reads are mapped beyond 200 nt into regions lacking enrichment probes. Dotted arrow: heterozygous mutation in parent 1; solid arrow: homozygous mutation in patient 1. (B) Conventional Sanger sequencing chromatograms show the distinct homozygous mutations (51G>A; 55G>A) and compound heterozygous mutations (30G>A and 111G>A) in MOPD I patients.

Fig. 1

Identification of RNU4ATAC mutations in MOPD I patients. (A) High throughput sequencing of ∼1.8 Mb mapped region. Top: Overview of enriched sequence reads from a single MOPD I patient, mapped to the target region showing chromosome 2 coordinates and schematic of genes. Bottom: Paired end reads mapped to targeted region, visualized using IGV browser (Broad Institute), showing the depth of sequencing coverage (gray peaks) at tiled enrichment probes (purple rectangles, bottom); see Supplementary Table 1. Very few sequence reads are mapped beyond 200 nt into regions lacking enrichment probes. Dotted arrow: heterozygous mutation in parent 1; solid arrow: homozygous mutation in patient 1. (B) Conventional Sanger sequencing chromatograms show the distinct homozygous mutations (51G>A; 55G>A) and compound heterozygous mutations (30G>A and 111G>A) in MOPD I patients.

Fig. 2

Mutations in RNU4ATAC affect U12-dependent spliceosomal function. (A) U4atac snRNA mutations found in MOPD I patients disrupt minor class intron splicing in vivo. Chinese Hamster Ovary cells were transfected with a test intron and various snRNA constructs (fig. S8) (10). After 48 hr, RNA splicing products were analyzed by RT-PCR, followed by agarose gel electrophoresis to determine U12-dependent minor spliceosome activities. Effects on splicing in vivo were quantitated relative to the wild type U4atac titration curve shown in figure S9 and plotted as the mean and standard deviation of triplicate transfections. Lane 1 contains no U4atac snRNA construct while lanes 2-8 contain U4atac-ATH constructs with the indicated sequence changes. (B) Endogenous U12-dependent introns are inefficiently spliced in MOPD I cells. The ratio of spliced to unspliced pre-mRNA for U12- and U2-dependent introns was determined using real time RT-PCR. Two MOPD I fibroblast cell lines homozygous for the 51G>A mutation were compared to two normal fibroblast cell lines. The spliced to unspliced ratio in the normal cells was set to unity for each intron. The horizontal lines show the group average for U12-dependent introns (average=0.45) and U2-dependent introns (average=1.01). The genes and introns examined are listed in table S2. (C) Restoration of wild type U4atac snRNA increases U12-dependent splicing in MOPD I cells. MOPD I fibroblasts were transfected with a wild type human U4atac snRNA gene driven by a U1 snRNA promoter or vector DNA alone. The same introns were measured for splicing as in panel B. The spliced to unspliced ratio of the cells transfected with vector alone was set to unity. The horizontal lines show the group average for U12-dependent introns (average=1.67) and U2-dependent introns (average=1.14).

Fig. 2

Mutations in RNU4ATAC affect U12-dependent spliceosomal function. (A) U4atac snRNA mutations found in MOPD I patients disrupt minor class intron splicing in vivo. Chinese Hamster Ovary cells were transfected with a test intron and various snRNA constructs (fig. S8) (10). After 48 hr, RNA splicing products were analyzed by RT-PCR, followed by agarose gel electrophoresis to determine U12-dependent minor spliceosome activities. Effects on splicing in vivo were quantitated relative to the wild type U4atac titration curve shown in figure S9 and plotted as the mean and standard deviation of triplicate transfections. Lane 1 contains no U4atac snRNA construct while lanes 2-8 contain U4atac-ATH constructs with the indicated sequence changes. (B) Endogenous U12-dependent introns are inefficiently spliced in MOPD I cells. The ratio of spliced to unspliced pre-mRNA for U12- and U2-dependent introns was determined using real time RT-PCR. Two MOPD I fibroblast cell lines homozygous for the 51G>A mutation were compared to two normal fibroblast cell lines. The spliced to unspliced ratio in the normal cells was set to unity for each intron. The horizontal lines show the group average for U12-dependent introns (average=0.45) and U2-dependent introns (average=1.01). The genes and introns examined are listed in table S2. (C) Restoration of wild type U4atac snRNA increases U12-dependent splicing in MOPD I cells. MOPD I fibroblasts were transfected with a wild type human U4atac snRNA gene driven by a U1 snRNA promoter or vector DNA alone. The same introns were measured for splicing as in panel B. The spliced to unspliced ratio of the cells transfected with vector alone was set to unity. The horizontal lines show the group average for U12-dependent introns (average=1.67) and U2-dependent introns (average=1.14).