Repetitive Exons Research Articles

TASOR is a pseudo-PARP that directs HUSH complex assembly and epigenetic transposon control

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits — TASOR, MPP8 and Periphilin — contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.

Evolution of the Highly Repetitive PEVK Region of Titin Across Mammals.

The protein titin plays a key role in vertebrate muscle where it acts like a giant molecular spring. Despite its importance and conservation over vertebrate evolution, a lack of high quality annotations in non-model species makes comparative evolutionary studies of titin challenging. The PEVK region of titin—named for its high proportion of Pro-Glu-Val-Lys amino acids—is particularly difficult to annotate due to its abundance of alternatively spliced isoforms and short, highly repetitive exons. To understand PEVK evolution across mammals, we developed a bioinformatics tool, PEVK_Finder, to annotate PEVK exons from genomic sequences of titin and applied it to a diverse set of mammals. PEVK_Finder consistently outperforms standard annotation tools across a broad range of conditions and improves annotations of the PEVK region in non-model mammalian species. We find that the PEVK region can be divided into two subregions (PEVK-N, PEVK-C) with distinct patterns of evolutionary constraint and divergence. The bipartite nature of the PEVK region has implications for titin diversification. In the PEVK-N region, certain exons are conserved and may be essential, but natural selection also acts on particular codons. In the PEVK-C, exons are more homogenous and length variation of the PEVK region may provide the raw material for evolutionary adaptation in titin function. The PEVK-C region can be further divided into a highly repetitive region (PEVK-CA) and one that is more variable (PEVK-CB). Taken together, we find that the very complexity that makes titin a challenge for annotation tools may also promote evolutionary adaptation.

Characterization of Circular RNA Concatemers.

Circular RNAs (circRNAs) constitute a novel subset in the fascinating world of noncoding RNA, and they are found in practically all eukaryotes. Most of them exhibit low expression levels but some are extremely abundant. Typically, circRNAs are studied by RT-PCR-based assays, but for certain types of analyses this technique is not suitable. Circular RNA with repetitive exons (circular concatemers) has been observed by us and others when transiently expressing circRNAs in cells, however techniques and assays to study these species have not been established. Here, this chapter outlines three biochemical assays (RNase R-, RNase H-, and alkaline-treatment) that combined with northern blotting are useful to study circRNAs in general and circular RNA concatemers in particular.

Novel dentin phosphoprotein frameshift mutations in dentinogenesis imperfecta type II

The dentin sialophosphoprotein (DSPP) gene encodes the most abundant non-collagenous protein in tooth dentin and DSPP protein is cleaved into several segments including the highly phosphorylated dentin phosphoprotein (DPP). Mutations in the DSPP gene have been solely related to non-syndromic form of hereditary dentin defects. We recruited three Korean families with dentinogenesis imperfecta (DGI) type II and sequenced the exons and exon-intron boundaries of the DSPP gene based on the candidate gene approach. Direct sequencing of PCR products and allele-specific cloning of the highly repetitive exon 5 revealed novel single base pair (bp) deletional mutations (c.2688delT and c.3560delG) introducing hydrophobic amino acids in the hydrophilic repeat domain of the DPP coding region. All affected members of the three families showed exceptionally rapid pulp chambers obliteration, even before tooth eruption. Individuals with the c.3560delG mutation showed only mild, yellowish tooth discoloration, in contrast to the affected individuals from two families with c.2688delT mutation. We believe that these results will help us to understand the molecular pathogenesis of DGI type II as well as the normal process of dentin biomineralization.

Molecular characterization of the porcine deleted in malignant brain tumors 1 gene ( DMBT1)

The human gene deleted in malignant brain tumors 1 ( DMBT1) is considered to play a role in tumorigenesis and pathogen defense. It encodes a protein with multiple scavenger receptor cysteine-rich (SRCR) domains, which are involved in recognition and binding of a broad spectrum of bacterial pathogens. The SRCR domains are encoded by highly homologous repetitive exons, whose number in humans may vary from 8 to 13 due to genetic polymorphism. Here, we characterized the porcine DMBT1 gene on the mRNA and genomic level. We assembled a 4.5 kb porcine DMBT1 cDNA sequence from RT-PCR amplified seminal vesicle RNA. The porcine DMBT1 cDNA contains an open reading frame of 4050 nt. The transcript gives rise to a putative polypeptide of 1349 amino acids with a calculated mass of 147.9 kDa. Compared to human DMBT1, it contains only four N-terminal SRCR domains. Northern blotting revealed transcripts of ∼ 4.7 kb in size in the tissues analyzed. Analysis of ESTs suggested the existence of secreted and transmembrane variants. The porcine DMBT1 gene spans about 54 kb on chromosome 14q28-q29. In contrast to the characterized cDNA, the genomic BAC clone only contained 3 exons coding for N-terminal SRCR domains. In different mammalian DMBT1 orthologs large interspecific differences in the number of SRCR exons and utilization of the transmembrane exon exist. Our data suggest that the porcine DMBT1 gene may share with the human DMBT1 gene additional intraspecific variations in the number of SRCR-coding exons.

Population Haplotypes of Exon ORF15 of the Retinitis Pigmentosa GTPase Regulator Gene in Germany

Mutations in exon ORF15 of the retinitis pigmentosa GTPase regulator gene (RPGR) within chromosomal region Xp21.1 are a significant cause of a number of retinal disorders. The high mutation rate is ascribed to the highly repetitive, purine-rich tracts within the exon ORF15 sequence. Importantly, all exon ORF15 mutations observed to date represent protein-truncating mutations (nonsense and frameshift mutations). Because of its repetitive motifs, mutation screening of the hot-spot region by direct DNA sequencing is a technically challenging task. We devised a screening strategy for exon ORF15 mutations that reserves DNA sequencing for precise sizing and base-order assessment of detected mutations. The screening strategy is based on a PCR/restriction fragment length polymorphism (RFLP) analysis of exon ORF15 and comparison with population-specific RFLP haplotypes. The latter were constructed from PCR/RFLP analysis of DNA samples from 100 healthy German male individuals. Mutational alterations of normal RFLP haplotype patterns were predicted. Six distinct RFLP haplotypes (founder alleles H1-H6) were observed with frequencies ranging from 2% to 63%. All natural variations of exon ORF15 were in-frame alterations ranging in size between 3bp and 36bp. Prediction of mutation-specific RFLP patterns indicated a high detection rate of mutations. A new strategy has been developed using routine protocols for mutation screening of difficult-to-sequence, highly repetitive exon ORF15 of the RPGR gene in a German population.

Mutational risk in highly repetitive exon ORF15 of the RPGR multidisease gene is not associated with haplotype background

Exon ORF15 is an alternative exon in the retinitis pigmentosa GTPase regulator (RPGR) gene containing a highly repetitive, purine-rich internal region. It constitutes a mutational hot spot giving rise to a group of heterogeneous X-linked retinal disorders. We sought to determine whether non-pathogenic substitutions and sequence length variations in the repetitive sequence have an influence on the risk of pathogenic exon ORF15 mutations. The type and distribution of exon ORF15 polymorphisms were assessed by genotyping 107 healthy German males using standard procedures. Polymorphisms were grouped into haplotypes and their frequencies determined in normal controls and previously analyzed patients with X-linked retinitis pigmentosa (XLRP). In the control group we identified 6 complex variants of the most common, ancestral exon ORF15 allele corresponding to the GenBank reference sequence (accession no. AF286472). Exon ORF15 mutations in XLRP patients were associated with the ancestral allele in 75% of affected cases. Four of the most recent founder haplotypes termed H3, H4, H6 and H7 were not identified in the patient samples. Our analysis and review of polymorphism data from the published literature suggests the presence of common exon ORF15 haplotypes in the European population. While the mutational risk in the RPGR gene appears not to be altered by the haplotype background, exon ORF15 haplotype analysis may be useful for tracing the evolutionary history of RP3-associated diseases.

Fiber-FISH analysis of the 3'-terminal region of the human L-type Ca2+ channel alpha 1C subunit gene.

Human L-type Ca2+ channel alpha 1C subunit gene (CACNL1A1) maps to the distal region of chromosome 12p13, and is composed of approximately 50 exons spanning over 150 kb of the human genome as estimated by restriction map analysis. However, the structure and the total length of the 3'-end of the gene is not clear because the size of several big introns remains unknown. Here the fiber-FISH technique was used to determine the relative order and size of eight partial genomic DNA clones from the central and 3'-terminal regions of CACNL1A1. The total physical distance of this region, including the size and gap distances between the clones were re-estimated. The results show that the physical order of the tested clones was 5'-g14-5 > g12-2 > g10-8 > g4-5 > g16-7 > g8-3 > g12-5 > g6-20-3'. Their individual sizes vary between 6.7 and 21.9 kb. Clones g6-20 and g12-5, both containing repetitive exon 45/46-like element, were found to be located within 59.1 kb downstream of g8-3 containing earlier identified polyadenylation site, i.e. 229.5 kb away from clone g14-5 (exons 10, 11). The possible implications of this structural complexity is discussed.

Cloning and expression of the human gene for transaldolase. A novel highly repetitive element constitutes an integral part of the coding sequence.

A novel highly repetitive retrotransposable element was cloned based on a limited sequence homology to the human T-cell leukemia virus and a related endogenous retroviral sequence, HRES-1. This repetitive element was found to constitute an integral part of the coding sequence of the human gene for transaldolase. In comparison with the intronless yeast gene, structural analysis of the human transaldolase genomic locus revealed that the human gene is comprised of five exons, second and third of which uniquely developed by insertion of a retrotransposable element. The 1329-base pair full-length cDNA, clone 4/2-4/1, contains an open reading frame coding for a protein of 336 amino acids with a predicted molecular mass of 38 kDa. This protein shows a 58% overall sequence homology with the 37-kDa yeast transaldolase. Antibodies raised against a 22-kDa recombinant polypeptide expressed from a 474-base pair 5' fragment of clone 4/2-4/1, containing repetitive exons 2 and 3, cross-reacted with yeast transaldolase and recognized the 38-kDa native human protein. Detection of a retrotransposon in the coding sequence of the human transaldolase gene demonstrates the importance of these repetitive elements in evolution of the eukaryotic genome.