The prevalent c.903+469T>C mutation in MTRR causes the cblE type of homocystinuria by strengthening an SRSF1 binding site in an ESE leading to activation of a pseudoexon. We hypothesized that other splicing regulatory elements (SREs) are also critical for MTRR pseudoexon inclusion. We demonstrate that the MTRR pseudoexon is on the verge of being recognized and is therefore vulnerable to several point mutations that disrupt a fine-tuned balance between the different SREs. Normally, pseudoexon inclusion is suppressed by a hnRNP A1 binding exonic splicing silencer (ESS). When the c.903+469T>C mutation is present two ESEs abrogate the activity of the ESS and promote pseudoexon inclusion. Blocking the 3'splice site or the ESEs by SSOs is effective in restoring normal splicing of minigenes and endogenous MTRR transcripts in patient cells. By employing an SSO complementary to both ESEs, we were able to rescue MTRR enzymatic activity in patient cells to approximately 50% of that in controls. We show that several point mutations, individually, can activate a pseudoexon, illustrating that this mechanism can occur more frequently than previously expected. Moreover, we demonstrate that SSO blocking of critical ESEs is a promising strategy to treat the increasing number of activated pseudoexons.

• MeSH
• buněčné linie MeSH
• exony * MeSH
• ferredoxin-NADP-reduktasa genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• homocystinurie enzymologie   genetika   MeSH
• kultivované buňky MeSH
• lidé MeSH
• megaloblastová anemie enzymologie   genetika   MeSH
• místa sestřihu RNA MeSH
• mutace * MeSH
• oligonukleotidy * MeSH
• regulační sekvence ribonukleových kyselin * MeSH
• sestřih RNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Analysis of plants bearing a T-DNA insertion is a potent tool of modern molecular biology, providing valuable information about the function and involvement of genes in metabolic pathways. A collection of 12 Arabidopsis thaliana lines with T-DNA insertions in the gene coding for the catalytic subunit of telomerase (AtTERT) and in adjacent regions was screened for telomerase activity [telomere repeat amplification protocol (TRAP) assay], telomere length (terminal restriction fragments), and AtTERT transcription (quantitative reverse transcription-PCR). Lines with the insertion located upstream of the start codon displayed unchanged telomere stability and telomerase activity, defining a putative minimal AtTERT promoter and the presence of a regulatory element linked to increased transcription in the line SALK_048471. Lines bearing a T-DNA insertion inside the protein-coding region showed telomere shortening and lack of telomerase activity. Transcription in most of these lines was unchanged upstream of the T-DNA insertion, while it was notably decreased downstream. The expression profile varied markedly in mutant lines harbouring insertions at the 5' end of AtTERT which showed increased transcription and abolished tissue specificity. Moreover, the line FLAG_385G01 (T-DNA insertion inside intron 1) revealed the presence of a highly abundant downstream transcript with normal splicing but without active telomerase. The role of regulatory elements found along the AtTERT gene is discussed in respect to natural telomerase expression and putative intron-mediated enhancement.

• MeSH
• Arabidopsis genetika   MeSH
• DNA bakterií genetika   MeSH
• genotyp MeSH
• inzerční mutageneze MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proteiny huseníčku genetika   MeSH
• regulace genové exprese u rostlin MeSH
• regulační oblasti nukleových kyselin genetika   MeSH
• telomerasa genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Splicing-affecting mutations can disrupt gene function by altering the transcript assembly. To ascertain splicing dysregulation principles, we modified a minigene assay for the parallel high-throughput evaluation of different mutations by next-generation sequencing. In our model system, all exonic and six intronic positions of the SMN1 gene's exon 7 were mutated to all possible nucleotide variants, which amounted to 180 unique single-nucleotide mutants and 470 double mutants. The mutations resulted in a wide range of splicing aberrations. Exonic splicing-affecting mutations resulted either in substantial exon skipping, supposedly driven by predicted exonic splicing silencer or cryptic donor splice site (5'ss) and de novo 5'ss strengthening and use. On the other hand, a single disruption of exonic splicing enhancer was not sufficient to cause major exon skipping, suggesting these elements can be substituted during exon recognition. While disrupting the acceptor splice site led only to exon skipping, some 5'ss mutations potentiated the use of three different cryptic 5'ss. Generally, single mutations supporting cryptic 5'ss use displayed better pre-mRNA/U1 snRNA duplex stability and increased splicing regulatory element strength across the original 5'ss. Analyzing double mutants supported the predominating splicing regulatory elements' effect, but U1 snRNA binding could contribute to the global balance of splicing isoforms. Based on these findings, we suggest that creating a new splicing enhancer across the mutated 5'ss can be one of the main factors driving cryptic 5'ss use.

• MeSH
• alternativní sestřih * MeSH
• buněčné linie MeSH
• exony * MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• místa sestřihu RNA MeSH
• mutace * MeSH
• mutageneze MeSH
• protein přežití motorických neuronů 1 chemie   genetika   metabolismus   MeSH
• RNA malá jaderná chemie   genetika   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• vazba proteinů MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

For more than three decades, researchers have known that consensus splice sites alone are not sufficient regulatory elements to provide complex splicing regulation. Other regulators, so-called splicing regulatory elements (SREs) are needed. Most importantly, their sequence variants often underlie the development of various human disorders. However, due to their variable location and high degeneracy, these regulatory sequences are also very difficult to recognize and predict. Many different approaches aiming to identify SREs have been tried, often leading to the development of in silico prediction tools. While these tools were initially expected to be helpful to identify splicing-affecting mutations in genetic diagnostics, we are still quite far from meeting this goal. In fact, most of these tools are not able to accurately discern the SRE-affecting pathological variants from those not affecting splicing. Nonetheless, several recent evaluations have given appealing results (namely for EX-SKIP, ESRseq and Hexplorer predictors). In this review, we aim to summarize the history of the different approaches to SRE prediction, and provide additional validation of these tools based on patients' clinical data. Finally, we evaluate their usefulness for diagnostic settings and discuss the challenges that have yet to be met.

• MeSH
• diagnostické techniky molekulární metody   trendy   MeSH
• genetické nemoci vrozené * MeSH
• lidé MeSH
• místa sestřihu RNA * MeSH
• mutace * MeSH
• prekurzory RNA genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Both variants affecting splice sites and those in splicing regulatory elements (SREs) can impair pre-mRNA splicing, eventually leading to severe diseases. Despite the availability of many prediction tools, prognosis of splicing affection is not trivial, especially when SREs are involved. Here, we present data on 92 in silico-/55 minigene-analysed variants detected in genes responsible for the primary immunodeficiencies development (namely BTK, CD40LG, IL2RG, SERPING1, STAT3, and WAS). Of 20 splicing-affecting variants, 16 affected splice site while 4 disrupted potential SRE. The presence or absence of splicing defects was confirmed in 30 of 32 blood-derived patients' RNAs. Testing prediction tools performance, splice site disruptions and creations were reliably predicted in contrast to SRE-affecting variants for which just ESRseq, ΔHZEI-scores and EX-SKIP predictions showed promising results. Next, we found an interesting pattern in cryptic splice site predictions. These results might help PID-diagnosticians and geneticists cope with potential splicing-affecting variants.

• MeSH
• buňky Hep G2 MeSH
• dítě MeSH
• exony MeSH
• HeLa buňky MeSH
• kojenec MeSH
• komplement C1 - inaktivátory genetika   MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• mutace MeSH
• předškolní dítě MeSH
• protein Wiskottova-Aldrichova syndromu genetika   MeSH
• receptory interleukinů - společná gama-podjednotka genetika   MeSH
• rekombinantní fúzní proteiny genetika   MeSH
• sestřih RNA * MeSH
• syndromy imunologické nedostatečnosti genetika   MeSH
• transkripční faktor STAT3 genetika   MeSH
• tyrosinkinasy genetika   MeSH
• U937 buňky MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH

Značná část lidských chorob má genetickou příčinu a pro úspěšnou léčbu je často nezbytné přesně určit, která mutace chorobu způsobuje. Identifikace kauzálních mutací však není vždy jednoznačná, a to i tehdy, máme-li k dispozici relevantní sekvence DNA. Moderní celogenomové metody odhalí řadu sekvenčních variant, u kterých není příčinná souvislost s chorobou zřejmá. Mnohé varianty ovlivňují genovou expresi změnou sestřihu pre-mRNA, ale tento efekt nemusí být odhalen, pokud nebylo již dříve prokázáno, že daná oblast obsahuje regulační elementy sestřihu (SRE). Jejich identifikace je proto klíčovým faktorem pro vyhodnocení vlivu takových mutací na vznik choroby. Projekt si klade za cíl lokalizovat funkční SRE a pokusit se nalézt obecná pravidla jejich výskytu v cílových sekvencích. Proto bude provedena systematická experimentální analýza vybraných oblastí genů, které jsou zodpovědné za vznik závažných lidských chorob. Získaná mapa míst náchylných k mutacím vyvolávajícím změny sestřihu bude využita jako pomocný nástroj pro diagnostiku aberantního sestřihu u nově identifikovaných mutací.; Significant proportion of human diseases is genetically determined and successful treatment is dependent on precise diagnosis of causal mutations. However, their identification is not always so unambiguous, although relevant sequence data is available. Modern genomic methods are able to uncover a considerable number of sequence variants with unknown impact on disease. Sequence variants frequently influence the gene expression through alteration of pre-mRNA splicing, but this effect can be hidden, if the splicing regulatory function of mutated regions was not proven in advance. The identification of splicing regulatory elements (SREs) is therefore a crucial step in evaluation of each mutation as potentially pathogenic. The aim of the project is to localize functional SREs and to try to define some rules for their occurrence by performing a systematic experimental analysis of ten regions in genes involved in disease development. Acquired „hot spot“ map of splicing affecting regions should act as an additional tool for diagnostics of aberrant splicing in newly identified gene variants.

• MeSH
• genetická predispozice k nemoci MeSH
• genetické nemoci vrozené genetika   MeSH
• lidé MeSH
• mutace genetika   MeSH
• sestřih RNA genetika   MeSH
• Check Tag
• lidé MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• genetika, lékařská genetika
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Mutations affecting splicing underlie the development of many human genetic diseases, but rather rarely through mechanisms of pseudoexon activation. Here, we describe a novel c.1092T>A mutation in the iduronate-2-sulfatase (IDS) gene detected in a patient with significantly decreased IDS activity and a clinical diagnosis of mild mucopolysaccharidosis II form. The mutation created an exonic de novo acceptor splice site and resulted in a complex splicing pattern with multiple pseudoexon activation in the patient's fibroblasts. Using an extensive series of minigene splicing experiments, we showed that the competition itself between the de novo and authentic splice site led to the bypass of the authentic one. This event then resulted in activation of several cryptic acceptor and donor sites in the upstream intron. As this was an unexpected and previously unreported mechanism of aberrant pseudoexon inclusion, we systematically analysed and disproved that the patient's mutation induced any relevant change in surrounding splicing regulatory elements. Interestingly, all pseudoexons included in the mature transcripts overlapped with the IDS alternative terminal exon 7b suggesting that this sequence represents a key element in the IDS pre-mRNA architecture. These findings extend the spectrum of mechanisms enabling pseudoexon activation and underscore the complexity of mutation-induced splicing aberrations. KEY MESSAGE: Novel exonic IDS gene mutation leads to a complex splicing pattern. Mutation activates multiple pseudoexons through a previously unreported mechanism. Multiple cryptic splice site (ss) activation results from a bypass of authentic ss. Authentic ss bypass is due to a competition between de novo and authentic ss.

• MeSH
• bodová mutace MeSH
• exony MeSH
• glykoproteiny genetika   MeSH
• introny MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• místa sestřihu RNA MeSH
• mladiství MeSH
• mukopolysacharidóza II genetika   MeSH
• mutace MeSH
• sestřih RNA MeSH
• Check Tag
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH

Non-coding RNAs (ncRNAs) are important regulatory molecules involved in various physiological and pathological cellular processes. Small nucleolar RNAs (snoRNAs), subclass of small ncRNAs, have been considered important but unglamorous elements in the production of protein synthesis machinery of cells. However, recent evidence has indicated that these non-coding RNAs might have a crucial role also in controlling cell behavior, and snoRNAs dysfunction could significantly contribute to carcinogenesis. Here, we summarize the most important aspects of snoRNAs biology, their functioning in cancer cell, and potential usage in diagnosis or as a new class of therapeutic targets in cancer.

• MeSH
• alternativní sestřih MeSH
• cílená molekulární terapie MeSH
• fyziologický stres MeSH
• lidé MeSH
• malá jadérková RNA fyziologie   MeSH
• nádorové biomarkery fyziologie   MeSH
• nádory genetika   metabolismus   terapie   MeSH
• posttranskripční úpravy RNA MeSH
• regulace genové exprese u nádorů MeSH
• RNA interference MeSH
• sekvence nukleotidů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Single nucleotide polymorphisms located in 5' untranslated regions (5'UTRs) can regulate gene expression and have clinical impact. Recognition of functionally significant sequences within 5'UTRs is crucial in next-generation sequencing applications. Furthermore, information about the behavior of 5'UTRs during gene evolution is scarce. Using the example of the ATP-binding cassette transporter A1 (ABCA1) gene (Tangier disease), we describe our algorithm for functionally significant sequence finding. 5'UTR features (upstream start and stop codons, open reading frames (ORFs), GC content, motifs, and secondary structures) were studied using freely available bioinformatics tools in 55 vertebrate orthologous genes obtained from Ensembl and UCSC. The most conserved sequences were suggested as hot spots. Exon and intron enhancers and silencers (sc35, ighg2 cgamma2, ctnt, gh-1, and fibronectin eda exon), transcription factors (TFIIA, TATA, NFAT1, NFAT4, and HOXA13), some of them cancer related, and microRNA (hsa-miR-4474-3p) were localized to these regions. An upstream ORF, overlapping with the main ORF in primates and possibly coding for a small bioactive peptide, was also detected. Moreover, we showed several features of 5'UTRs, such as GC content variation, hairpin structure conservation or 5'UTR segmentation, which are interesting from a phylogenetic point of view and can stimulate further evolutionary oriented research.

• MeSH
• 5' nepřekládaná oblast * genetika   MeSH
• ABCA1 protein chemie   genetika   MeSH
• anotace sekvence MeSH
• fylogeneze MeSH
• introny MeSH
• konformace nukleové kyseliny MeSH
• konzervovaná sekvence genetika   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• nukleotidové motivy genetika   MeSH
• otevřené čtecí rámce genetika   MeSH
• savci genetika   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sestřih RNA genetika   MeSH
• zastoupení bazí genetika   MeSH
• zesilovače transkripce genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH

p53 is one of the most studied tumor suppressor proteins that plays an important role in basic biological processes including cell cycle, DNA damage response, apoptosis, and senescence. The human TP53 gene contains alternative promoters that produce N-terminally truncated proteins and can produce several isoforms due to alternative splicing. p53 function is realized by binding to a specific DNA response element (RE), resulting in the transactivation of target genes. Here, we evaluated the influence of quadruplex DNA structure on the transactivation potential of full-length and N-terminal truncated p53α isoforms in a panel of S. cerevisiae luciferase reporter strains. Our results show that a G-quadruplex prone sequence is not sufficient for transcription activation by p53α isoforms, but the presence of this feature in proximity to a p53 RE leads to a significant reduction of transcriptional activity and changes the dynamics between co-expressed p53α isoforms.

• MeSH
• G-kvadruplexy * MeSH
• lidé MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• promotorové oblasti (genetika) genetika   MeSH
• protein - isoformy genetika   metabolismus   MeSH
• proteiny regulující apoptózu genetika   metabolismus   MeSH
• protoonkogenní proteiny genetika   metabolismus   MeSH
• responzivní elementy genetika   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH