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
• Názvy látek
• ketoglutarátdehydrogenasový komplex MeSH
• messenger RNA MeSH
• místa sestřihu RNA MeSH
• prekurzory RNA MeSH
• protein - isoformy MeSH
• sestřihové faktory MeSH
• vápník 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.

• Klíčová slova
• evaluation of prediction tools, in silico predictions, mutation, pre-mRNA splicing, splicing aberration, splicing regulatory elements, variants of unknown significance,
• 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
• Názvy látek
• místa sestřihu RNA * MeSH
• prekurzory RNA MeSH

Genetically encoded far-red and near-infrared fluorescent proteins enable efficient imaging in studies of tumorigenesis, embryogenesis, and inflammation in model animals. Here we report comparative testing of available GFP-like far-red fluorescent proteins along with a modified protein, named Katushka2S, and near-infrared bacterial phytochrome-based markers. We compare fluorescence signal and signal-to-noise ratio at various excitation wavelength and emission filter combinations using transiently transfected cell implants in mice, providing a basis for rational choice of optimal marker(s) for in vivo imaging studies. We demonstrate that the signals of various far-red fluorescent proteins can be spectrally unmixed based on different signal-to-noise ratios in different channels, providing the straightforward possibility of multiplexed imaging with standard equipment. Katushka2S produced the brightest and fastest maturing fluorescence in all experimental setups. At the same time, signal-to-noise ratios for Katushka2S and near-infrared bacterial phytochrome, iRFP720 were comparable in their optimal channels. Distinct spectral and genetic characteristics suggest this pair of a far-red and a near-infrared fluorescent protein as an optimal combination for dual color, whole body imaging studies in model animals.

• MeSH
• alternativní sestřih MeSH
• celotělové zobrazování * metody   MeSH
• červený fluorescenční protein MeSH
• HEK293 buňky MeSH
• heterografty MeSH
• lidé MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• místa sestřihu RNA MeSH
• modely u zvířat MeSH
• molekulární zobrazování metody   MeSH
• myši MeSH
• poměr signál - šum MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• luminescentní proteiny MeSH
• místa sestřihu RNA MeSH

Mutations in the first nucleotide of exons (E(+1)) mostly affect pre-mRNA splicing when found in AG-dependent 3' splice sites, whereas AG-independent splice sites are more resistant. The AG-dependency, however, may be difficult to assess just from primary sequence data as it depends on the quality of the polypyrimidine tract. For this reason, in silico prediction tools are commonly used to score 3' splice sites. In this study, we have assessed the ability of sequence features and in silico prediction tools to discriminate between the splicing-affecting and non-affecting E(+1) variants. For this purpose, we newly tested 16 substitutions in vitro and derived other variants from literature. Surprisingly, we found that in the presence of the substituting nucleotide, the quality of the polypyrimidine tract alone was not conclusive about its splicing fate. Rather, it was the identity of the substituting nucleotide that markedly influenced it. Among the computational tools tested, the best performance was achieved using the Maximum Entropy Model and Position-Specific Scoring Matrix. As a result of this study, we have now established preliminary discriminative cut-off values showing sensitivity up to 95% and specificity up to 90%. This is expected to improve our ability to detect splicing-affecting variants in a clinical genetic setting.

• MeSH
• agamaglobulinemie genetika   MeSH
• bodová mutace * MeSH
• exony MeSH
• genetické nemoci vázané na chromozom X genetika   MeSH
• HeLa buňky MeSH
• lidé MeSH
• místa sestřihu RNA * MeSH
• modely genetické MeSH
• molekulární sekvence - údaje MeSH
• počítačová simulace MeSH
• proteinkinasa BTK MeSH
• sekvenční analýza DNA MeSH
• sestřih RNA MeSH
• software * MeSH
• tyrosinkinasy genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• místa sestřihu RNA * MeSH
• proteinkinasa BTK MeSH
• tyrosinkinasy MeSH

Deep intronic mutations are often ignored as possible causes of human diseases. A deep intronic mutation in the MTRR gene, c.903+469T>C, is the most frequent mutation causing the cblE type of homocystinuria. It is well known to be associated with pre-mRNA mis-splicing, resulting in pseudoexon inclusion; however, the pathological mechanism remains unknown. We used minigenes to demonstrate that this mutation is the direct cause of MTRR pseudoexon inclusion, and that the pseudoexon is normally not recognized due to a suboptimal 5' splice site. Within the pseudoexon we identified an exonic splicing enhancer (ESE), which is activated by the mutation. Cotransfection and siRNA experiments showed that pseudoexon inclusion depends on the cellular amounts of SF2/ASF and in vitro RNA-binding assays showed dramatically increased SF2/ASF binding to the mutant MTRR ESE. The mutant MTRR ESE sequence is identical to an ESE of the alternatively spliced MST1R proto-oncogene, which suggests that this ESE could be frequently involved in splicing regulation. Our study conclusively demonstrates that an intronic single nucleotide change is sufficient to cause pseudoexon activation via creation of a functional ESE, which binds a specific splicing factor. We suggest that this mechanism may cause genetic disease much more frequently than previously reported.

• MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• exony genetika   MeSH
• ferredoxin-NADP-reduktasa genetika   MeSH
• homocystinurie klasifikace   enzymologie   genetika   MeSH
• introny genetika   MeSH
• jaderné proteiny metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• místa sestřihu RNA genetika   MeSH
• molekulární sekvence - údaje MeSH
• mutace genetika   MeSH
• mutantní proteiny genetika   MeSH
• proteiny vázající RNA metabolismus   MeSH
• protoonkogen Mas MeSH
• sekvence nukleotidů MeSH
• serin-arginin sestřihové faktory MeSH
• sestřih RNA genetika   MeSH
• vazba proteinů MeSH
• vitamin B 12 metabolismus   MeSH
• výpočetní biologie MeSH
• zesilovače transkripce genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ferredoxin-NADP-reduktasa MeSH
• jaderné proteiny MeSH
• MAS1 protein, human MeSH Prohlížeč
• messenger RNA MeSH
• methionine synthase reductase MeSH Prohlížeč
• místa sestřihu RNA MeSH
• mutantní proteiny MeSH
• proteiny vázající RNA MeSH
• protoonkogen Mas MeSH
• serin-arginin sestřihové faktory MeSH
• vitamin B 12 MeSH