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

U2AF65 (U2AF2) and PUF60 (PUF60) are splicing factors important for recruitment of the U2 small nuclear ribonucleoprotein to lariat branch points and selection of 3' splice sites (3'ss). Both proteins preferentially bind uridine-rich sequences upstream of 3'ss via their RNA recognition motifs (RRMs). Here, we examined 36 RRM substitutions reported in cancer patients to identify variants that alter 3'ss selection, RNA binding and protein properties. Employing PUF60- and U2AF65-dependent 3'ss previously identified by RNA-seq of depleted cells, we found that 43% (10/23) and 15% (2/13) of independent RRM mutations in U2AF65 and PUF60, respectively, conferred splicing defects. At least three RRM mutations increased skipping of internal U2AF2 (~9%, 2/23) or PUF60 (~8%, 1/13) exons, indicating that cancer-associated RRM mutations can have both cis- and trans-acting effects on splicing. We also report residues required for correct folding/stability of each protein and map functional RRM substitutions on to existing high-resolution structures of U2AF65 and PUF60. These results identify new RRM residues critical for 3'ss selection and provide relatively simple tools to detect clonal RRM mutations that enhance the mRNA isoform diversity.

• Publikační typ
• časopisecké články MeSH

PUF60 is a splicing factor that binds uridine (U)-rich tracts and facilitates association of the U2 small nuclear ribonucleoprotein with primary transcripts. PUF60 deficiency (PD) causes a developmental delay coupled with intellectual disability and spinal, cardiac, ocular and renal defects, but PD pathogenesis is not understood. Using RNA-Seq, we identify human PUF60-regulated exons and show that PUF60 preferentially acts as their activator. PUF60-activated internal exons are enriched for Us upstream of their 3' splice sites (3'ss), are preceded by longer AG dinucleotide exclusion zones and more distant branch sites, with a higher probability of unpaired interactions across a typical branch site location as compared to control exons. In contrast, PUF60-repressed exons show U-depletion with lower estimates of RNA single-strandedness. We also describe PUF60-regulated, alternatively spliced isoforms encoding other U-bound splicing factors, including PUF60 partners, suggesting that they are co-regulated in the cell, and identify PUF60-regulated exons derived from transposed elements. PD-associated amino-acid substitutions, even within a single RNA recognition motif (RRM), altered selection of competing 3'ss and branch points of a PUF60-dependent exon and the 3'ss choice was also influenced by alternative splicing of PUF60. Finally, we propose that differential distribution of RNA processing steps detected in cells lacking PUF60 and the PUF60-paralog RBM39 is due to the RBM39 RS domain interactions. Together, these results provide new insights into regulation of exon usage by the 3'ss organization and reveal that germline mutation heterogeneity in RRMs can enhance phenotypic variability at the level of splice-site and branch-site selection.

• MeSH
• aminokyselinové motivy MeSH
• exony * MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• heterogenní jaderné ribonukleoproteiny metabolismus   MeSH
• jaderné proteiny metabolismus   MeSH
• krátké rozptýlené jaderné elementy MeSH
• lidé MeSH
• malý jaderný ribonukleoprotein U1 metabolismus   MeSH
• missense mutace * MeSH
• místa sestřihu RNA * MeSH
• proteiny vázající RNA metabolismus   MeSH
• represorové proteiny chemie   nedostatek   metabolismus   MeSH
• sekvenční analýza RNA MeSH
• sestřihové faktory chemie   nedostatek   metabolismus   MeSH
• sestřihový faktor U2AF MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cílem projektu je definovat nepříznivé situace v komplexní léčbě karcinomu prsu žen, při kterých vzniká riziko léčbou indukovaného nárustu heterogenity čili divergence vlastností nádoru a doporučit způsob jak je monitorovat a předcházet jim.

• MeSH
• DNA nádorová MeSH
• hormonální protinádorové látky MeSH
• nádory prsu terapie   diagnóza   klasifikace   MeSH
• radioterapie MeSH

• Konspekt
• Lékařské vědy. Lékařství
• NLK Obory
• onkologie
• endokrinologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Heterozygosity for ataxia-telangiectasia (A-T), a cancer-prone recessive syndrome, has been associated with an increased risk of breast cancer. The gene for A-T (ATM) is located at chromosomal region 11q22-q23, a region of frequent loss of constitutional heterozygosity in breast and other tumors. Loss of constitutional heterozygosity at 1lq22-q23 was found in 47% of informative cases in the series of primary tumors analyzed in this study. To investigate the role of ATM in breast cancer, we have determined the complete genomic organization of the gene, developed an exon-scanning PCR single-strand conformation polymorphism (PCR-SSCP) assay for mutation detection of ATM, and screened 38 consecutive breast tumors for mutations using both genomic DNA- and cDNA-based assays. In addition to common ATM polymorphisms detected both in the coding sequence and in flanking introns, seven unique SSCP alleles were identified in six tumor DNAs. Sequence analysis of these alleles revealed rive nucleotide substitutions that were predicted to change the encoded amino acid. However, PCR-SSCP and nucleotide sequencing analysis of the paired blood samples and of an extended sample size of a total of 224 chromosomes indicated that these SSCP patterns represent constitutional rare polymorphisms with a frequency between 0.005 and 0.023. Because the majority of A-T mutations are null mutations and none of the ATM alleles found in breast cancer samples would lead to the truncation of the translation product, we conclude that, in this initial sample of sporadic breast cancer patients, there was no evidence for an increased number of A-T carriers. In addition, because no somatic mutations were found, our study rules out the ATM gene as the frequently altered tumor suppressor gene at 11q23.

• MeSH
• delece genu * MeSH
• lidé středního věku MeSH
• lidé MeSH
• lidské chromozomy, pár 11 * genetika   MeSH
• molekulární sekvence - údaje MeSH
• mutační analýza DNA MeSH
• náchylnost k nemoci MeSH
• nádory prsu * genetika   MeSH
• polymerázová řetězová reakce MeSH
• sekvence nukleotidů MeSH
• teleangiektatická ataxie * genetika   MeSH
• tumor supresorové geny * MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH