Germline CHEK2 pathogenic variants confer an increased risk of female breast cancer (FBC). Here we describe a recurrent germline intronic variant c.1009-118_1009-87delinsC, which showed a splice acceptor shift in RNA analysis, introducing a premature stop codon (p.Tyr337PhefsTer37). The variant was found in 21/10,204 (0.21%) Czech FBC patients compared to 1/3250 (0.03%) controls (p = 0.04) and in 4/3639 (0.11%) FBC patients from an independent German dataset. In addition, we found this variant in 5/2966 (0.17%) Czech (but none of the 443 German) ovarian cancer patients, three of whom developed early-onset tumors. Based on these observations, we classified this variant as likely pathogenic.
- MeSH
- checkpoint kinasa 2 * genetika MeSH
- dospělí MeSH
- genetická predispozice k nemoci * genetika MeSH
- introny * genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádory prsu * genetika MeSH
- nádory vaječníků genetika MeSH
- prekurzory RNA genetika MeSH
- sestřih RNA * genetika MeSH
- zárodečné mutace * MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
- Německo MeSH
Protein tyrosine phosphatase, nonreceptor type 22 (PTPN22), is an archetypal non-HLA autoimmunity gene. It is one of the most prominent genetic contributors to type 1 diabetes mellitus outside the HLA region, and prevalence of its risk variants is subject to enormous geographic variability. Here, we address the genetic background of patients with type 1 diabetes mellitus of Armenian descent. Armenia has a population that has been genetically isolated for 3000 years. We hypothesized that two PTPN22 polymorphisms, rs2476601 and rs1310182, are associated with type 1 diabetes mellitus in persons of Armenian descent. In this association study, we genotyped the allelic frequencies of two risk-associated PTPN22 variants in 96 patients with type 1 diabetes mellitus and 100 controls of Armenian descent. We subsequently examined the associations of PTPN22 variants with the manifestation of type 1 diabetes mellitus and its clinical characteristics. We found that the rs2476601 minor allele (c.1858T) frequency in the control population was very low (q = 0.015), and the trend toward increased frequency of c.1858CT heterozygotes among patients with type 1 diabetes mellitus was not significant (OR 3.34, 95% CI 0.88-12.75; χ2 test p > 0.05). The control population had a high frequency of the minor allele of rs1310182 (q = 0.375). The frequency of c.2054-852TC heterozygotes was significantly higher among the patients with type 1 diabetes mellitus (OR 2.39, 95% CI 1.35-4.24; χ2 test p < 0.001), as was the frequency of the T allele (OR 4.82, 95% CI 2.38-9.76; χ2 test p < 0.001). The rs2476601 c.1858CT genotype and the T allele correlated negatively with the insulin dose needed three to six months after diagnosis. The rs1310182 c.2054-852CC genotype was positively associated with higher HbA1c at diagnosis and 12 months after diagnosis. We have provided the first information on diabetes-associated polymorphisms in PTPN22 in a genetically isolated Armenian population. We found only a limited contribution of the prototypic gain-of-function PTPN22 polymorphism rs2476601. In contrast, we found an unexpectedly close association of type 1 diabetes mellitus with rs1310182.
- MeSH
- diabetes mellitus 1. typu * genetika MeSH
- fosfatasy MeSH
- introny MeSH
- lidé MeSH
- polymorfismus genetický MeSH
- tyrosinfosfatasa nereceptorového typu 22 genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Arménie MeSH
Among alternative splicing events in the human transcriptome, tandem NAGNAG acceptor splice sites represent an appreciable proportion. Both proximal and distal NAG can be used to produce two splicing isoforms differing by three nucleotides. In some cases, the upstream exon can be alternatively spliced as well, which further increases the number of possible transcripts. In this study, we showed that NAG choice in tandem splice site depends considerably not only on the concerned acceptor, but also on the upstream donor splice site sequence. Using an extensive set of experiments with systematically modified two-exonic minigene systems of AFAP1L2 or CSTD gene, we recognized the third and fifth intronic upstream donor splice site position and the tandem acceptor splice site region spanning from -10 to +2, including NAGNAG itself, as the main drivers. In addition, competition between different branch points and their composition were also shown to play a significant role in NAG choice. All these nucleotide effects appeared almost additive, which explained the high variability in proximal versus distal NAG usage.
Ca2+-insensitive and -sensitive E1 subunits of the 2-oxoglutarate dehydrogenase complex (OGDHC) regulate tissue-specific NADH and ATP supply by mutually exclusive OGDH exons 4a and 4b. Here we show that their splicing is enforced by distant lariat branch points (dBPs) located near the 5' splice site of the intervening intron. dBPs restrict the intron length and prevent transposon insertions, which can introduce or eliminate dBP competitors. The size restriction was imposed by a single dominant dBP in anamniotes that expanded into a conserved constellation of four dBP adenines in amniotes. The amniote clusters exhibit taxon-specific usage of individual dBPs, reflecting accessibility of their extended motifs within a stable RNA hairpin rather than U2 snRNA:dBP base-pairing. The dBP expansion took place in early terrestrial species and was followed by a uridine enrichment of large downstream polypyrimidine tracts in mammals. The dBP-protected megatracts permit reciprocal regulation of exon 4a and 4b by uridine-binding proteins, including TIA-1/TIAR and PUF60, which promote U1 and U2 snRNP recruitment to the 5' splice site and BP, respectively, but do not significantly alter the relative dBP usage. We further show that codons for residues critically contributing to protein binding sites for Ca2+ and other divalent metals confer the exon inclusion order that mirrors the Irving-Williams affinity series, linking the evolution of auxiliary splicing motifs in exons to metallome constraints. Finally, we hypothesize that the dBP-driven selection for Ca2+-dependent ATP provision by E1 facilitated evolution of endothermy by optimizing the aerobic scope in target tissues.
- MeSH
- alternativní sestřih * MeSH
- exony MeSH
- HEK293 buňky MeSH
- introny * MeSH
- ketoglutarátdehydrogenasový komplex genetika metabolismus MeSH
- lidé MeSH
- messenger RNA chemie metabolismus MeSH
- místa sestřihu RNA MeSH
- molekulární evoluce MeSH
- obratlovci genetika MeSH
- prekurzory RNA chemie metabolismus MeSH
- protein - isoformy genetika metabolismus MeSH
- rozptýlené repetitivní sekvence MeSH
- sestřihové faktory metabolismus MeSH
- spliceozomy metabolismus MeSH
- termoregulace genetika MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Chromatin architect of muscle expression (Charme) is a muscle-restricted long noncoding RNA (lncRNA) that plays an important role in myogenesis. Earlier evidence indicates that the nuclear Charme isoform, named pCharme, acts on the chromatin by assisting the formation of chromatin domains where myogenic transcription occurs. By combining RNA antisense purification (RAP) with mass spectrometry and loss-of-function analyses, we have now identified the proteins that assist these chromatin activities. These proteins-which include a sub-set of splicing regulators, principally PTBP1 and the multifunctional RNA/DNA binding protein MATR3-bind to sequences located within the alternatively spliced intron-1 to form nuclear aggregates. Consistent with the functional importance of pCharme interactome in vivo, a targeted deletion of the intron-1 by a CRISPR-Cas9 approach in mouse causes the release of pCharme from the chromatin and results in cardiac defects similar to what was observed upon knockout of the full-length transcript.
- MeSH
- heterogenní jaderné ribonukleoproteiny metabolismus MeSH
- introny genetika MeSH
- lidé MeSH
- myši MeSH
- protein vázající polypyrimidinové úseky RNA metabolismus MeSH
- proteiny asociované s jadernou matrix metabolismus MeSH
- proteiny vázající RNA metabolismus MeSH
- RNA dlouhá nekódující metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The 12 members of the ABCA subfamily in humans are known for their ability to transport cholesterol and its derivatives, vitamins, and xenobiotics across biomembranes. Several ABCA genes are causatively linked to inborn diseases, and the role in cancer progression and metastasis is studied intensively. The regulation of translation initiation is implicated as the major mechanism in the processes of post-transcriptional modifications determining final protein levels. In the current bioinformatics study, we mapped the features of the 5' untranslated regions (5'UTR) known to have the potential to regulate translation, such as the length of 5'UTRs, upstream ATG codons, upstream open-reading frames, introns, RNA G-quadruplex-forming sequences, stem loops, and Kozak consensus motifs, in the DNA sequences of all members of the subfamily. Subsequently, the conservation of the features, correlations among them, ribosome profiling data as well as protein levels in normal human tissues were examined. The 5'UTRs of ABCA genes contain above-average numbers of upstream ATGs, open-reading frames and introns, as well as conserved ones, and these elements probably play important biological roles in this subfamily, unlike RG4s. Although we found significant correlations among the features, we did not find any correlation between the numbers of 5'UTR features and protein tissue distribution and expression scores. We showed the existence of single nucleotide variants in relation to the 5'UTR features experimentally in a cohort of 105 breast cancer patients. 5'UTR features presumably prepare a complex playground, in which the other elements such as RNA binding proteins and non-coding RNAs play the major role in the fine-tuning of protein expression.
- MeSH
- 5' nepřekládaná oblast genetika MeSH
- ABC transportér, podrodina A klasifikace genetika metabolismus MeSH
- biologický transport genetika MeSH
- cholesterol metabolismus MeSH
- introny genetika MeSH
- jednonukleotidový polymorfismus genetika MeSH
- lidé MeSH
- multigenová rodina genetika MeSH
- otevřené čtecí rámce genetika MeSH
- proteosyntéza genetika MeSH
- ribozomy genetika metabolismus MeSH
- výpočetní biologie MeSH
- xenobiotika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The universal nine-amino-acid transactivation domains (9aaTADs) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in all nine members of the specificity protein (SP) family. Previously, the Sp1 transcription factor has been defined as a glutamine-rich activator. We showed by amino acid substitutions that the glutamine residues are completely dispensable for 9aaTAD function and are not conserved in the SP family. We described the origin and evolutionary history of 9aaTADs. The 9aaTADs of the ancestral Sp2 gene became inactivated in early chordates. We next discovered that an accumulation of valines in 9aaTADs inactivated their transactivation function and enabled their strict conservation during evolution. Subsequently, in chordates, Sp2 has duplicated and created new paralogs, Sp1, Sp3, and Sp4 (the SP1-4 clade). During chordate evolution, the dormancy of the Sp2 activation domain lasted over 100 million years. The dormant but still intact ancestral Sp2 activation domains allowed diversification of the SP1-4 clade into activators and repressors. By valine substitution in the 9aaTADs, Sp1 and Sp3 regained their original activator function found in ancestral lower metazoan sea sponges. Therefore, the vertebrate SP1-4 clade could include both repressors and activators. Furthermore, we identified secondary 9aaTADs in Sp2 introns present from fish to primates, including humans. In the gibbon genome, introns containing 9aaTADs were used as exons, which turned the Sp2 gene into an activator. Similarly, we identified introns containing 9aaTADs used conditionally as exons in the (SP family-unrelated) transcription factor SREBP1, suggesting that the intron-9aaTAD reservoir is a general phenomenon.
- MeSH
- aktivace transkripce MeSH
- duplikace genu MeSH
- fylogeneze MeSH
- introny * genetika MeSH
- lidé MeSH
- molekulární evoluce * MeSH
- regulace genové exprese * MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie MeSH
- transkripční faktor Sp2 * antagonisté a inhibitory genetika metabolismus MeSH
- valin genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
Acceptor splice site recognition (3' splice site: 3'ss) is a fundamental step in precursor messenger RNA (pre-mRNA) splicing. Generally, the U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor (U2AF) heterodimer recognizes the 3'ss, of which U2AF35 has a dual function: (i) It binds to the intron-exon border of some 3'ss and (ii) mediates enhancer-binding splicing activators' interactions with the spliceosome. Alternative mechanisms for 3'ss recognition have been suggested, yet they are still not thoroughly understood. Here, we analyzed 3'ss recognition where the intron-exon border is bound by a ubiquitous splicing regulator SRSF1. Using the minigene analysis of two model exons and their mutants, BRCA2 exon 12 and VARS2 exon 17, we showed that the exon inclusion correlated much better with the predicted SRSF1 affinity than 3'ss quality, which were assessed using the Catalog of Inferred Sequence Binding Preferences of RNA binding proteins (CISBP-RNA) database and maximum entropy algorithm (MaxEnt) predictor and the U2AF35 consensus matrix, respectively. RNA affinity purification proved SRSF1 binding to the model 3'ss. On the other hand, knockdown experiments revealed that U2AF35 also plays a role in these exons' inclusion. Most probably, both factors stochastically bind the 3'ss, supporting exon recognition, more apparently in VARS2 exon 17. Identifying splicing activators as 3'ss recognition factors is crucial for both a basic understanding of splicing regulation and human genetic diagnostics when assessing variants' effects on splicing.
- MeSH
- alternativní sestřih genetika MeSH
- exony genetika MeSH
- HeLa buňky MeSH
- introny genetika MeSH
- lidé MeSH
- místa sestřihu RNA genetika fyziologie MeSH
- proteiny vázající RNA metabolismus MeSH
- regulační oblasti nukleových kyselin genetika MeSH
- sekvence nukleotidů genetika MeSH
- serin-arginin sestřihové faktory metabolismus MeSH
- sestřih RNA fyziologie MeSH
- sestřihové faktory metabolismus fyziologie MeSH
- sestřihový faktor U2AF metabolismus MeSH
- spliceozomy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The universal nine-amino-acid transactivation domains (9aaTADs) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in all nine members of the specificity protein (SP) family. Previously, the Sp1 transcription factor has been defined as a glutamine-rich activator. We showed by amino acid substitutions that the glutamine residues are completely dispensable for 9aaTAD function and are not conserved in the SP family. We described the origin and evolutionary history of 9aaTADs. The 9aaTADs of the ancestral Sp2 gene became inactivated in early chordates. We next discovered that an accumulation of valines in 9aaTADs inactivated their transactivation function and enabled their strict conservation during evolution. Subsequently, in chordates, Sp2 has duplicated and created new paralogs, Sp1, Sp3, and Sp4 (the SP1-4 clade). During chordate evolution, the dormancy of the Sp2 activation domain lasted over 100 million years. The dormant but still intact ancestral Sp2 activation domains allowed diversification of the SP1-4 clade into activators and repressors. By valine substitution in the 9aaTADs, Sp1 and Sp3 regained their original activator function found in ancestral lower metazoan sea sponges. Therefore, the vertebrate SP1-4 clade could include both repressors and activators. Furthermore, we identified secondary 9aaTADs in Sp2 introns present from fish to primates, including humans. In the gibbon genome, introns containing 9aaTADs were used as exons, which turned the Sp2 gene into an activator. Similarly, we identified introns containing 9aaTADs used conditionally as exons in the (SP family-unrelated) transcription factor SREBP1, suggesting that the intron-9aaTAD reservoir is a general phenomenon.
- MeSH
- aktivace transkripce MeSH
- duplikace genu MeSH
- fylogeneze MeSH
- introny genetika MeSH
- lidé MeSH
- molekulární evoluce * MeSH
- regulace genové exprese * MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie MeSH
- transkripční faktor Sp2 antagonisté a inhibitory genetika metabolismus MeSH
- valin genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
PURPOSE: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. METHODS: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. RESULTS: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. CONCLUSION: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.
- MeSH
- ABC transportéry genetika MeSH
- genomika MeSH
- introny MeSH
- lidé MeSH
- makulární degenerace * genetika MeSH
- mutace MeSH
- rodokmen MeSH
- Stargardtova nemoc MeSH
- transkriptom * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
