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

• MeSH
• eukaryotické buňky fyziologie   MeSH
• genetická transkripce * fyziologie   MeSH
• posttranskripční úpravy RNA fyziologie   MeSH
• prekurzory RNA fyziologie   MeSH
• prokaryotické buňky fyziologie   MeSH
• RNA heterogenní jaderná fyziologie   MeSH

Ribosome biogenesis is an essential, energy demanding process whose deregulation has been implicated in cancer, aging, and neurodegeneration. Ribosome biogenesis is therefore under surveillance of pathways including the p53 tumor suppressor. Here, we first performed a high-content siRNA-based screen of 175 human ribosome biogenesis factors, searching for impact on p53. Knock-down of 4 and 35 of these proteins in U2OS cells reduced and increased p53 abundance, respectively, including p53 accumulation after depletion of BYSL, DDX56, and WDR75, the effects of which were validated in several models. Using complementary approaches including subcellular fractionation, we demonstrate that endogenous human WDR75 is a nucleolar protein and immunofluorescence analysis of ectopic GFP-tagged WDR75 shows relocation to nucleolar caps under chemically induced nucleolar stress, along with several canonical nucleolar proteins. Mechanistically, we show that WDR75 is required for pre-rRNA transcription, through supporting the maintenance of physiological levels of RPA194, a key subunit of the RNA polymerase I. Furthermore, WDR75 depletion activated the RPL5/RPL11-dependent p53 stabilization checkpoint, ultimately leading to impaired proliferation and cellular senescence. These findings reveal a crucial positive role of WDR75 in ribosome biogenesis and provide a resource of human ribosomal factors the malfunction of which affects p53.

• MeSH
• buněčné jadérko genetika   metabolismus   MeSH
• DEAD-box RNA-helikasy metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• molekuly buněčné adheze metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• prekurzory RNA metabolismus   MeSH
• ribozomální proteiny * genetika   metabolismus   MeSH
• ribozomy genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Clinical management that enables efficient cancer prevention in carriers of germ-line alterations in cancer susceptibility genes requires and unequivocal characterization of the pathogenic mutation. Current cancer predisposition analyses reveal many variants of unknown clinical significance (VUS) which complicate the clinical interpretation of results. The VUS that change splicing of corresponding gene product may be considered as pathogenic. Our project aims to identify splicing alterations in carriers of VUS in genes predisposing to breast and ovarian cancer (CHEK2, PALB2, NBN, TP53, RAD51C/D, MLH1, MSH2/6, BRIP1). First, we aim to perform a comprehensive characterization of naturally occurring splicing variants of analyzed genes in normal lymphocytes, mammary and adipose tissues from non-cancer individuals using multiplex (m)PCR/next generation sequencing (NGS) analysis. Subsequently, the analyses of RNA from carriers of VUS will enable to characterize the qualitative (fragment analysis, mPCR/NGS) and quantitative (qPCR) changes in splicing pattern of corresponding genes.

Prevence vzniku nádorů u nosičů dědičných alterací nádorových predispozičních genů vyžaduje jasnou identifikaci zodpovědných patogenních mutací. Při analýze nádorové predispozice je odhalena řada variant s nejasným významem (VUS) komplikujících klinickou interpretaci výsledků. Pokud VUS postihují proces sestřihu transkriptu příslušného genu, lze je považovat za patogenní. V projektu se zaměříme na vyhodnocení změn sestřihového vzorce mRNA transkriptů u nosičů dědičných variant genů predisponujících ke vzniku dědičné formy karcinomu (ca) prsu a ovarií (CHEK2, PALB2, NBN, TP53, RAD51C/D, MLH1, MSH2/6, BRIP1). Nejprve provedeme identifikaci jejich sestřihových variant v normálních tkáních (leukocyty, mamární a tuková tkáň) zdravých osob bez přítomnosti dědičných variant pomocí multiplexní PCR (mPCR) umožňující amplifikaci všech sestřihových oblastí s následnou charakterizací sekvenovánín nové generace (NGS). Vyšetření RNA od nosičů VUS umožní kvalitativně (fragmentační a mPCR/NGS analýza) i kvantitativně (qPCR) identifikovat VUS způsobující změny sestřihového vzorce příslušného genu.

• MeSH
• dědičný syndrom nádoru prsu a vaječníků genetika   klasifikace   MeSH
• genetická predispozice k nemoci genetika   klasifikace   MeSH
• geny nádorové genetika   MeSH
• prekurzory RNA MeSH
• sestřih RNA genetika   MeSH
• sestřihové faktory MeSH
• vysoce účinné nukleotidové sekvenování MeSH

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

RNA splicing, the process of intron removal from pre-mRNA, is essential for the regulation of gene expression. It is controlled by the spliceosome, a megadalton RNA-protein complex that assembles de novo on each pre-mRNA intron through an ordered assembly of intermediate complexes1,2. Spliceosome activation is a major control step that requires substantial protein and RNA rearrangements leading to a catalytically active complex1-5. Splicing factor 3B subunit 1 (SF3B1) protein-a subunit of the U2 small nuclear ribonucleoprotein6-is phosphorylated during spliceosome activation7-10, but the kinase that is responsible has not been identified. Here we show that cyclin-dependent kinase 11 (CDK11) associates with SF3B1 and phosphorylates threonine residues at its N terminus during spliceosome activation. The phosphorylation is important for the association between SF3B1 and U5 and U6 snRNAs in the activated spliceosome, termed the Bact complex, and the phosphorylation can be blocked by OTS964, a potent and selective inhibitor of CDK11. Inhibition of CDK11 prevents spliceosomal transition from the precatalytic complex B to the activated complex Bact and leads to widespread intron retention and accumulation of non-functional spliceosomes on pre-mRNAs and chromatin. We demonstrate a central role of CDK11 in spliceosome assembly and splicing regulation and characterize OTS964 as a highly selective CDK11 inhibitor that suppresses spliceosome activation and splicing.

• MeSH
• aktivace enzymů účinky léků   MeSH
• chinolony farmakologie   MeSH
• chromatin metabolismus   MeSH
• cyklin-dependentní kinasy * antagonisté a inhibitory   metabolismus   MeSH
• fosfoproteiny * chemie   metabolismus   MeSH
• fosforylace MeSH
• malý jaderný ribonukleoprotein U2 * chemie   metabolismus   MeSH
• prekurzory RNA * genetika   metabolismus   MeSH
• sestřih RNA * účinky léků   MeSH
• spliceozomy * účinky léků   metabolismus   MeSH
• threonin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

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

Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus (Flaviviridae), is a causative agent of a severe neuroinfection. Recently, several flaviviruses have been shown to interact with host protein synthesis. In order to determine whether TBEV interacts with this host process in its natural target cells, we analysed de novo protein synthesis in a human cell line derived from cerebellar medulloblastoma (DAOY HTB-186). We observed a significant decrease in the rate of host protein synthesis, including the housekeeping genes HPRT1 and GAPDH and the known interferon-stimulated gene viperin. In addition, TBEV infection resulted in a specific decrease of RNA polymerase I (POLR1) transcripts, 18S and 28S rRNAs and their precursor, 45-47S pre-rRNA, but had no effect on the POLR3 transcribed 5S rRNA levels. To our knowledge, this is the first report of flavivirus-induced decrease of specifically POLR1 rRNA transcripts accompanied by host translational shut-off.

• MeSH
• genetická transkripce MeSH
• klíšťová encefalitida genetika   metabolismus   virologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• prekurzory RNA MeSH
• proteosyntéza genetika   MeSH
• RNA ribozomální genetika   metabolismus   MeSH
• RNA-polymerasa I genetika   metabolismus   MeSH
• viry klíšťové encefalitidy fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Purpose: To report molecular genetic findings in six probands with congenital hereditary endothelial dystrophy (CHED) variably associated with hearing loss (also known as Harboyan syndrome). Furthermore, we developed a cellular model to determine if disease-associated variants induce aberrant SLC4A11 pre-mRNA splicing. Methods: Direct sequencing of the entire SLC4A11 coding region was performed in five probands. In one individual, whole genome sequencing was undertaken. The effect of c.2240+5G>A on pre-mRNA splicing was evaluated in a corneal endothelial-like (CE-like) cell model expressing SLC4A11. CE-like cells were derived from autologous induced pluripotent stem cells (iPSCs) via neural crest cells exposed to B27, PDGF-BB, and DKK-2. Total RNA was extracted, and RT-PCR was performed followed by Sanger and a targeted next generation sequencing (NGS) approach to identify and quantify the relative abundance of alternatively spliced transcripts. Results: In total, 11 different mutations in SLC4A11 evaluated as pathogenic were identified; of these, c.1237G>A, c.2003T>C, c.1216+1G>A, and c.2240+5G>A were novel. The c.2240+5G>A variant was demonstrated to result in aberrant pre-mRNA splicing. A targeted NGS approach confirmed that the variant introduces a leaky cryptic splice donor site leading to the production of a transcript containing an insertion of six base pairs with the subsequent introduction of a premature stop codon (p.Thr747*). Furthermore, a subset of transcripts comprising full retention of intron 16 also were observed, leading to the same functionally null allele. Conclusions: This proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants.

• MeSH
• antiportéry biosyntéza   genetika   MeSH
• buněčná diferenciace MeSH
• dědičné dystrofie rohovky genetika   metabolismus   patologie   MeSH
• dítě MeSH
• dospělí MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• kultivované buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• percepční nedoslýchavost genetika   metabolismus   patologie   MeSH
• předškolní dítě MeSH
• prekurzory RNA MeSH
• proteiny přenášející anionty biosyntéza   genetika   MeSH
• regulace genové exprese * MeSH
• RNA genetika   MeSH
• rodokmen MeSH
• rohovkový endotel metabolismus   patologie   MeSH
• senioři MeSH
• sestřih RNA MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cellular mechanisms that safeguard genome integrity are often subverted in cancer. To identify cancer-related genome caretakers, we employed a convergent multi-screening strategy coupled to quantitative image-based cytometry and ranked candidate genes according to multivariate readouts reflecting viability, proliferative capacity, replisome integrity, and DNA damage signaling. This unveiled regulators of replication stress resilience, including components of the pre-mRNA cleavage and polyadenylation complex. We show that deregulation of pre-mRNA cleavage impairs replication fork speed and leads to excessive origin activity, rendering cells highly dependent on ATR function. While excessive formation of RNA:DNA hybrids under these conditions was tightly associated with replication-stress-induced DNA damage, inhibition of transcription rescued fork speed, origin activation, and alleviated replication catastrophe. Uncoupling of pre-mRNA cleavage from co-transcriptional processing and export also protected cells from replication-stress-associated DNA damage, suggesting that pre-mRNA cleavage provides a mechanism to efficiently release nascent transcripts and thereby prevent gene gating-associated genomic instability.

• MeSH
• aktivní transport - buněčné jádro MeSH
• DNA nádorová genetika   metabolismus   MeSH
• HeLa buňky MeSH
• heteroduplexy nukleové kyseliny genetika   metabolismus   MeSH
• jaderné proteiny genetika   metabolismus   MeSH
• lidé MeSH
• messenger RNA biosyntéza   genetika   MeSH
• nádory genetika   metabolismus   MeSH
• nestabilita genomu * MeSH
• polyadenylace MeSH
• poškození DNA * MeSH
• prekurzory RNA biosyntéza   genetika   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• regulace genové exprese u nádorů MeSH
• replikace DNA * MeSH
• RNA nádorová biosyntéza   genetika   MeSH
• štěpení RNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Split gene architecture of most human genes requires removal of intervening sequences by mRNA splicing that occurs on large multiprotein complexes called spliceosomes. Mutations compromising several spliceosomal components have been recorded in degenerative syndromes and haematological neoplasia, thereby highlighting the importance of accurate splicing execution in homeostasis of assorted adult tissues. Moreover, insufficient splicing underlies defective development of craniofacial skeleton and upper extremities. This review summarizes recent advances in the understanding of splicing factor function deduced from cryo-EM structures. We combine these data with the characterization of splicing factors implicated in hereditary or somatic disorders, with a focus on potential functional consequences the mutations may elicit in spliceosome assembly and/or performance. Given aberrant splicing or perturbations in splicing efficiency substantially underpin disease pathogenesis, profound understanding of the mis-splicing principles may open new therapeutic vistas. In three major sections dedicated to retinal dystrophies, hereditary acrofacial syndromes, and haematological malignancies, we delineate the noticeable variety of conditions associated with dysfunctional splicing and accentuate recurrent patterns in splicing defects.

• MeSH
• elektronová kryomikroskopie MeSH
• konformace proteinů MeSH
• lidé MeSH
• mutace MeSH
• nemoc genetika   MeSH
• prekurzory RNA genetika   MeSH
• ribonukleoproteiny malé jaderné chemie   genetika   ultrastruktura   MeSH
• sestřih RNA * MeSH
• spliceozomy genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH