Nuclear factor TDP-43 binds to the polymorphic TG repeats in CFTR intron 8 and causes skipping of exon 9: a functional link with disease penetrance.
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ABSTRACT:
SUBMITTER: Buratti E
PROVIDER: S-EPMC1182100 | biostudies-literature | 2004 Jun
REPOSITORIES: biostudies-literature
Publications
Nuclear factor TDP-43 binds to the polymorphic TG repeats in CFTR intron 8 and causes skipping of exon 9: a functional link with disease penetrance.
Buratti Emanuele E Brindisi Antonia A Pagani Franco F Baralle Francisco E FE
American journal of human genetics 20040601 6
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Project description:The putative role of (TG/CA)n repeats in the regulation of transcription has recently been reported for several cancer- and disease-related genes, including the genes encoding the epidermal growth factor receptor (EGFR), hydroxysteroid (11-beta) dehydrogenase 2 (HSD11B2) and interferon-gamma (IFNG). These studies indicated a correlation between gene expression levels and the presence or length of (TG/CA)n repeats in their intron 1 regions. A genome-wide search for genes with similar features may provide evidence of whether these dinucleotide repeats represent a class of universal regulators of gene expression, which has recently begun to be investigated as a quantitative complex phenotype. Using a public database of simple repeats, we identified 330 genes containing potentially polymorphic long (TG/CA)n repeats (n >or= 12) in their intron 1 regions. One known physiological pathway, the calcium signalling pathway, was found to be enriched among the genes containing long repeats. In addition, certain biological processes, such as cation transport, signal transduction and ion transport, were found to be enriched in these genes. Genotyping of the long repeats showed that the majority of these dinucleotide repeats were polymorphic in the HapMap CEU (Caucasians from Utah, USA) samples of northern and western European ancestry. Evidence for a significant association between these repeats and gene expression was not observed in the genes selected based on their expression profiles in the HapMap CEU samples. Our current findings, therefore, do not support a role for these repeats as a class of universal gene expression regulators. A more comprehensive evaluation of the relationship between these repeats and gene expression, potentially in other tissues, may be necessary to illustrate their roles in gene regulation in the future.
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Project description:TDP-43 is a nuclear RNA-binding protein that forms neuronal cytoplasmic inclusions in two major neurodegenerative diseases, ALS and FTLD. While the self-assembly of TDP-43 by its structured N-terminal and intrinsically disordered C-terminal domains has been widely studied, the mechanism by which mRNA preserves TDP-43 solubility in the nucleus has not been addressed. Here, we demonstrate that tandem RNA recognition motifs of TDP-43 bind to long GU-repeats in a cooperative manner through intermolecular interactions. Moreover, using mutants whose cooperativity is impaired, we found that the cooperative binding of TDP-43 to mRNA may be critical to maintain the solubility of TDP-43 in the nucleus and the miscibility of TDP-43 in cytoplasmic stress granules. We anticipate that the knowledge of a higher order assembly of TDP-43 on mRNA may clarify its role in intron processing and provide a means of interfering with the cytoplasmic aggregation of TDP-43.
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Project description:Spliceosomal twin introns (stwintrons) are introns where any of the three consensus sequences involved in splicing is interrupted by another intron (internal intron). In Aspergillus nidulans, a donor-disrupted stwintron (intron-1) is extant in the transcript encoding a reticulon-like protein. The orthologous transcript of Aspergillus niger can be alternatively spliced; the exon downstream the stwintron could be skipped by excising a sequence that comprises this stwintron, the neighbouring intron-2, and the exon bounded by these. This process involves the use of alternative 3' splice sites for the internal intron, the resulting alternative intervening sequence being a longer 3'-extended stwintron. In 29 species of Onygenales, a multi-step splicing process occurs in the orthologous transcript, in which a complex intervening sequence including the stwintron and neigbouring intron-2, generates by three splicing reactions a "second order intron" which must then be excised with a fourth splicing event. The gene model in two species can be envisaged as one canonical intron (intron-1) evolved from this complex intervening sequence of nested canonical introns found elsewhere in Onygenales. Postulated splicing intermediates were experimentally verified in one or more species. This work illustrates a role of stwintrons in both alternative splicing and the evolution of intron structure.
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Project description:PurposeTo evaluate the significance of molecular detection of cystic fibrosis transmembrane conductance regulator (CFTR) M470V, intron 8 poly-T, and intron 8 TG-repeats in congenital bilateral absence of the vas deferens (CBAVD).MethodsEighty-nine male patients with CBAVD and 103 healthy males were included in this study. Polymerase chain reaction was performed to amplify the polymorphic regions using primers from conserved regions. M470V was genotyped using real-time PCR by cycling probe. The exon 9 DNA sequence was determined using an automated sequencer. TG-repeats and poly-T were identified by direct sequencing analysis.ResultsThe 5T allele distribution was 0.32, 0.66 for 7T, and 0.02 for 9T in CBAVD males, respectively. In contrast, the 5T allele distribution was 0.03, 0.96 for 7T, and 0.01 for 9T in healthy control. Study of the polymorphisms of the upstream of exon 9 revealed a higher frequency of 5T allele in the CBAVD males. All cases with TG13T5 haplotype and TG12T5 homozygous led to CBAVD. The CFTR TG12T5-V470 variant haplotype was associated with CBAVD.ConclusionThe 5T allele of intron 8 of CFTR has clinically significant association with CBAVD. TG13T5 and TG12T5 homozygously led to CBAVD, and TG12T5-V470 may also lead to CBAVD.
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Project description:Abnormal accumulation of TDP-43 into cytoplasmic or nuclear inclusions with accompanying nuclear clearance, a common pathology initially identified in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), has also been found in Alzheimer' disease (AD). TDP-43 serves as a splicing repressor of nonconserved cryptic exons and that such function is compromised in brains of ALS and FTD patients, suggesting that nuclear clearance of TDP-43 underlies its inability to repress cryptic exons. However, whether TDP-43 cytoplasmic aggregates are a prerequisite for the incorporation of cryptic exons is not known. Here, we assessed hippocampal tissues from 34 human postmortem brains including cases with confirmed diagnosis of AD neuropathologic changes along with age-matched controls. We found that cryptic exon incorporation occurred in all AD cases exhibiting TDP-43 pathology. Furthermore, incorporation of cryptic exons was observed in the hippocampus when TDP-43 inclusions was restricted only to the amygdala, the earliest stage of TDP-43 progression. Importantly, cryptic exon incorporation could be detected in AD brains lacking TDP-43 inclusion but exhibiting nuclear clearance of TDP-43. These data supports the notion that the functional consequence of nuclear depletion of TDP-43 as determined by cryptic exon incorporation likely occurs as an early event of TDP-43 proteinopathy and may have greater contribution to the pathogenesis of AD than currently appreciated. Early detection and effective repression of cryptic exons in AD patients may offer important diagnostic and therapeutic implications for this devastating illness of the elderly.
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Project description:The DNA and RNA processing protein TDP-43 undergoes both functional and pathogenic aggregation. Functional TDP-43 aggregates form reversible, transient species such as nuclear bodies, stress granules, and myo-granules. Pathogenic, irreversible TDP-43 aggregates form in amyotrophic lateral sclerosis and other neurodegenerative conditions. Here we find the features of TDP-43 fibrils that confer both reversibility and irreversibility by determining structures of two segments reported to be the pathogenic cores of human TDP-43 aggregation: SegA (residues 311-360), which forms three polymorphs, all with dagger-shaped folds; and SegB A315E (residues 286-331 containing the amyotrophic lateral sclerosis hereditary mutation A315E), which forms R-shaped folds. Energetic analysis suggests that the dagger-shaped polymorphs represent irreversible fibril structures, whereas the SegB polymorph may participate in both reversible and irreversible fibrils. Our structures reveal the polymorphic nature of TDP-43 and suggest how the A315E mutation converts the R-shaped polymorph to an irreversible form that enhances pathology.
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Project description:Biallelic pathogenic variants in CFTR manifest as cystic fibrosis (CF) or other CFTR-related disorders (CFTR-RDs). The 5T allele, causing alternative splicing and reduced protein activity, is modulated by the adjacent TG repeat element, though previous data have been limited to small, selective cohorts. Here, the risk and spectrum of phenotypes associated with the CFTR TG-T5 haplotype variants (TG11T5, TG12T5, and TG13T5) in the absence of the p.Arg117His variant are evaluated. Individuals who received physician-ordered next-generation sequencing of CFTR were included. TG[11-13]T5 variant frequencies (biallelic or with another CF-causing variant [CFvar]) were calculated. Clinical information reported by the ordering provider or the individual was examined. Among 548,300 individuals, the T5 minor allele frequency (MAF) was 4.2% (TG repeat distribution: TG11 = 68.1%, TG12 = 29.5%, TG13 = 2.4%). When present with a CFvar, each TG[11-13]T5 variant was significantly enriched in individuals with a high suspicion of CF or CFTR-RD (personal/family history of CF/CFTR-RD) compared to those with a low suspicion for CF or CFTR-RD (hereditary cancer screening, CFTR not requisitioned). Compared to CFvar/CFvar individuals, those with TG[11-13]T5/CFvar generally had single-organ involvement, milder symptoms, variable expressivity, and reduced penetrance. These data improve our understanding of disease risks associated with TG[11-13]T5 variants and have important implications for reproductive genetic counseling.
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Project description:The molecular basis of the skipping of constitutive exons in many messenger RNAs is not fully understood. A well-studied example is exon 9 of the human cystic fibrosis transmembrane conductance regulator gene (CFTR), in which an abbreviated polypyrimidine tract between the branch point A and the 3' splice site is associated with increased exon skipping and disease. However, many exons, both in CFTR and in other genes and have short polypyrimidine tracts in their 3' splice sites, yet they are not skipped. Inspection of the 5' splice sites immediately up- and downstream of exon 9 revealed deviations from consensus sequence, so we hypothesized that this exon may be inherently vulnerable to skipping. To test this idea, we constructed a CFTR minigene and replicated exon 9 skipping associated with the length of the polypyrimidine tract upstream of exon 9. We then mutated the flanking 5' splice sites and determined the effect on exon skipping. Conversion of the upstream 5' splice site to consensus by replacing a pyrimidine at position +3 with a purine resulted in increased exon skipping. In contrast, conversion of the downstream 5' splice site to consensus by insertion of an adenine at position +4 resulted in a substantial reduction in exon 9 skipping, regardless of whether the upstream 5' splice site was consensus or not. These results suggested that the native downstream 5' splice site plays an important role in CFTR exon 9 skipping, a hypothesis that was supported by data from sheep and mouse genomes. Although CFTR exon 9 in sheep is preceded by a long polypyrimidine tract (Y(14)), it skips exon 9 in vivo and has a nonconsensus downstream 5' splice site identical to that in humans. On the other hand, CFTR exon 9 in mice is preceded by a short polypyrimidine tract (Y(5)) but is not skipped in vivo. Its downstream 5' splice site differs from that in humans by a 2-nt insertion, which, when introduced into the human CFTR minigene, abolished exon 9 skipping. Taken together, these observations place renewed emphasis on deviations at 5' splice sites in nucleotides other than the invariant GT, particularly when such changes are found in conjunction with other altered splicing sequences, such as a shortened polypyrimidine tract. Thus, careful inspection of entire 5' splice sites may identify constitutive exons that are vulnerable to skipping.
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