Mutations in U4atac snRNA, a Component of the Minor Spliceosome, in the Developmental Disorder MOPD I

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Science  08 Apr 2011:
Vol. 332, Issue 6026, pp. 238-240
DOI: 10.1126/science.1200587


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.

The small nuclear RNA (snRNA) U4atac is a component of the minor spliceosome and is required for the proper excision of the U12-dependent class of introns (14). Although they account for only about 800 introns in humans, U12-dependent introns are found in many essential genes, such as those involved in DNA replication and repair, transcription, and RNA processing and translation (5). Thus, mutations in an snRNA required to splice such introns are likely deleterious and presumably would result in important developmental or clinical consequences.

U4atac snRNA appears to be encoded by a single gene (RNU4ATAC) on chromosome 2q14.2. Here we report that biallelic mutations in this gene are found in a severe developmental disorder, microcephalic osteodysplastic primordial dwarfism type I (MOPD I; also known as Taybi-Linder syndrome, OMIM 210710) (6). The main features of MOPD I patients are extreme intrauterine growth retardation, abnormalities in multiple organs, and death in infancy or early childhood (7, 8). We focused our initial studies on cases diagnosed in the Amish of Ohio (9) (fig. S1), where uniform phenotypic features and a high degree of consanguinity suggested the existence of a single founder mutation. Briefly, we applied genome-wide homozygosity mapping followed by targeted, high-throughput second-generation sequencing in search of mutations at chromosome 2q14.2 (Fig. 1 and figs. S2 to S4). A novel g.51G>A variant within the non–protein-coding RNU4ATAC gene was detected in homozygosity in all seven Amish patients studied and in heterozygosity in 13 Amish parents. An Australian patient had the same mutation, whereas in two German MOPD 1 families, biallelic g.55G>A occurred in one patient and compound heterozygous g.30G>A and g.111G>A occurred in another patient. The 51G>A mutation represents a founder event in the Amish, as shown by haplotype analysis (figs. S3 and S5). This mutation was found in 16 of 281 Ohio Amish controls but in none of 180 Pennsylvania Amish controls. It was also seen in two of 720 controls from central Ohio but in none of 370 controls from France. The three mutations found in German MOPD 1 families were not found in 452 central Ohio controls. We conclude that the genetic findings are fully compatible with the expected recessive inheritance of rare mutations in the same gene [see (9) for further details of the mapping, sequencing, mutation analyses, and haplotyping].

Fig. 1

Identification of RNU4ATAC mutations in MOPD I patients. (A) High-throughput sequencing of mapped region (~1.8 Mb). 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 table S1. Very few sequence reads are mapped beyond 200 nucleotides 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.

The 30G>A, 51G>A, and 55G>A mutations in the U4atac snRNA are located within an important structural feature known as the 5′ stem-loop; the 111G>A mutation is located in another essential stem region, the 3′ stem-loop (10). These mutations are predicted to disrupt the snRNA’s secondary structure and cause defects in the minor spliceosome (fig. S6) (1113). To evaluate the functional effects of these mutations, we assayed the in vivo splicing of a modified U12-dependent intron reporter whose splicing is dependent on expression of a modified, exogenous U4atac snRNA, denoted U4atac-ATH (figs. S7 and S8) (10). Relative to wild-type U4atac, each of the MOPD I mutations in U4atac snRNA reduced U12-dependent splicing activity by greater than 90% (Fig. 2A). This suggests that all four mutations cause severe defects in U12-dependent splicing. Most of the splicing defect of the 51G>A mutation could be rescued by combining it with the 32C>T mutation in the presumed base-pairing partner (Fig. 2A, lane 8, and fig. S6). This suggests that the MOPD I mutation abrogates U4atac snRNA function by disrupting the RNA secondary structure.

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 hours, 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 fig. S9 and plotted as the mean and SD of triplicate transfections. Lane 1 contains no U4atac snRNA construct; lanes 2 to 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 by 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 (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).

U12-dependent introns are conserved in a variety of gene families implicated in different physiological processes (5, 14). To assess U12-dependent splicing in MOPD I cells, we compared fibroblasts obtained from two MOPD I patients (with the 51G>A mutation) and two normal human fibroblast cell lines. Using real-time reverse transcription polymerase chain reaction (RT-PCR) to quantify the levels of spliced and unspliced U12-dependent and U2-dependent introns, we found that all examined U12-dependent introns were less efficiently spliced in the MOPD I cells, whereas U2-dependent introns were not affected (Fig. 2B). Expression of wild-type U4atac snRNA in the MOPD I cells increased splicing of U12-dependent introns while having little effect on U2-dependent introns (Fig. 2C). Note that introns 5 and 12, which are the most affected (Fig. 2B), also showed the greatest increase upon restoration of wild-type U4atac snRNA (Fig. 2C). We also observed considerable variability in the reduction of splicing of individual U12-dependent introns in patient cells (Fig. 2B). Such variability may underlie the specific developmental defects seen in MOPD I. Similar variability has been observed in Drosophila carrying a mutation in U6atac snRNA (15).

These results are fully compatible with the idea that the MOPD I mutations in U4atac snRNA reduce in vivo splicing of U12-dependent introns, with potentially deleterious consequences for proper levels of gene expression and/or alternative splicing. Our findings illustrate the critical role of the minor spliceosome in human development. Future work to define the downstream affected genes in MOPD I patients is warranted.

Several observations suggest that normal function of the minor spliceosome is crucial for viability and development (16, 17). Other human diseases caused by mutations in protein components of small nuclear ribonucleoprotein (snRNP) complexes that contribute to spliceosome functions have been described (18, 19). We found genetic mutations in the U4atac snRNA gene by high-throughput sequencing of the entire mapped genomic locus, notably including non–protein-coding DNA. Much of the current emphasis in finding the mutations underlying Mendelian disorders is focused on exome sequencing, which would not have revealed the MOPD I mutations. This illustrates the need to sequence the genome rather than the exome in many situations.

Establishing a clinical diagnosis of MOPD (I, II, and III), Seckel syndrome, and related disorders has been difficult until now because of a considerable overlap in their clinical features (8, 20). In one study, mutations in the pericentrin (PCNT) gene were reported in patients with MOPD II but were not seen in patients with MOPD I or III, Seckel syndrome, or unclassified growth retardation (21). By contrast, other studies showed mutations in PCNT in patients with Seckel syndrome or MOPD II (2224). Even among the cases reported here, there were clear differences in the phenotypic features and life spans. We anticipate that further characterization of distinct mutations in RNU4ATAC will shed light on some of these difficulties of clinical classification.

Supporting Online Material

Materials and Methods

SOM Text

Figs. S1 to S9

Tables S1 to S3


References and Notes

  1. See supporting material on Science Online.
  2. We thank P. Mehlen and M.-M. Coissieux for the French control sample set, P. Meinecke for information on patients, J. Palatini for sharing equipment and advice, J. Lockman for technical support, and the Microarray Shared Resource and Nucleic Acid Shared Resource of the OSU Comprehensive Cancer Center for genotyping and sequencing. Supported by Ohio Supercomputing Center grant PAS0425-2 for computational resources (K.A. and D.E.S.), Leonard Krieger Fund of the Cleveland Foundation grant L2009-0078 (H.W.), NIH grants GM093074 and GM079527 (R.A.P.), National Cancer Institute grant P30 CA16058 (H.H., S.L., K.A., J.L., R.N., W.L., N.S., B.W., P.Y., H.W., D.E.S., J.A.W., and A.dlC.), and the OSU Comprehensive Cancer Center.
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