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The number of people involved in regular exercise and sports is increasing. Not infrequently, this is associated with intake of sports biologically active food supplements (BAS) and stimulators of physical ability. Data has been reported on the frequency of use of physical ability stimulators among professional athletes and on the use of the most popular food supplements among young people. Special attention is paid to the effect of such use on the cardiovascular system of athletes. This review describes negative cardiac effects and clinical cases of death of athletes due to the use of such supplements and stimulators.

Electronic supplementary material is available online at https://doi.org/10.6084/m9.figshare.c.5713034. Copyright © 2021 The Authors. Amplification of the proto-oncogene MYCN is a key molecular aberration in high-risk neuroblastoma and predictive of poor outcome in this childhood malignancy. We investigated the role of MYCN in regulating the protein cargo of extracellular vesicles (EVs) secreted by tumour cells that can be internalized by recipient cells with functional consequences. Using a switchable MYCN system coupled to mass spectrometry analysis, we found that MYCN regulates distinct sets of proteins in the EVs secreted by neuroblastoma cells. EVs produced by MYCN-expressing cells or isolated from neuroblastoma patients induced the Warburg effect, proliferation and c-MYC expression in target cells. Mechanistically, we linked the cancer-promoting activity of EVs to the glycolytic kinase pyruvate kinase M2 (PKM2) that was enriched in EVs secreted by MYC-expressing neuroblastoma cells. Importantly, the glycolytic enzymes PKM2 and hexokinase II were detected in the EVs circulating in the bloodstream of neuroblastoma patients, but not in those of non-cancer children. We conclude that MYC-activated cancers might spread oncogenic signals to remote body locations through EVs. Neuroblastoma UK to AS and FAPESP SPRINT Award (50356-4); FAPESP (2014/06863-3, 2018/18257-1, 2018/15549-1, 16/50356-4); CNPq “bolsa de produtividade”; Ricerca Finalizzata GR11-172. https://doi.org/10.6084/m9.figshare.c.5713034

Structural variation and different bioactivity of ionic liquids (ILs) make them highly promising for the development of novel biocides. Application of computational methods to the evaluation of potential antibacterial activity of chemical compounds is a useful, time- and cost-saving tool replacing numerous experimental syntheses. In the present study, quantitative structure-activity relationship (QSAR) modeling is applied to develop models (based on more than 800 data points) aiming to predict the minimal inhibitory concentration (MIC) of ILs against three types of human pathogens - Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The random forest model with the AlvaDesc descriptors in general demonstrates the best performance for all the three types of bacteria and is suggested as a final model. To interpret the final model and determine the most significant descriptors, a SHapley Additive exPlanation (SHAP) method was applied. Six amino acid ILs, which were synthesized for the first time, and five halogenide ionic liquids purchased, all based on 1-alkyl-3methylimidozolium cations with different alkyl chain lengths, C

Hutchinson–Gilford progeria syndrome (HGPS) is a rare, premature ageing syndrome in children. HGPS is normally caused by a mutation in the LMNA gene, encoding nuclear lamin A. The classical mutation in HGPS leads to the production of a toxic truncated version of lamin A, progerin, which retains a farnesyl group. Farnesyltransferase inhibitors (FTI), pravastatin and zoledronic acid have been used in clinical trials to target the mevalonate pathway in HGPS patients to inhibit farnesylation of progerin, in order to reduce its toxicity. Some other compounds that have been suggested as treatments include rapamycin, IGF1 and N-acetyl cysteine (NAC). We have analysed the distribution of prelamin A, lamin A, lamin A/C, progerin, lamin B1 and B2 in nuclei of HGPS cells before and after treatments with these drugs, an FTI and a geranylgeranyltransferase inhibitor (GGTI) and FTI with pravastatin and zoledronic acid in combination. Confirming other studies prelamin A, lamin A, progerin and lamin B2 staining was different between control and HGPS fibroblasts. The drugs that reduced progerin staining were FTI, pravastatin, zoledronic acid and rapamycin. However, drugs affecting the mevalonate pathway increased prelamin A, with only FTI reducing internal prelamin A foci. The distribution of lamin A in HGPS cells was improved with treatments of FTI, pravastatin and FTI + GGTI. All treatments reduced the number of cells displaying internal speckles of lamin A/C and lamin B2. Drugs targeting the mevalonate pathway worked best for progerin reduction, with zoledronic acid removing internal progerin speckles. Rapamycin and NAC, which impact on the MTOR pathway, both reduced both pools of progerin without increasing prelamin A in HGPS cell nuclei. Electronic supplementary material The online version of this article (10.1007/s10522-019-09807-4) contains supplementary material, which is available to authorized users.

Aim To demonstrate the immediate and medium-term results of the surgical treatment of patients with first metatarsophalangeal (MTP) joint osteoarthritis (OA) using the autologous matrix-induced chondrogenesis (AMIC) technique and to evaluate the effectiveness. Materials and Methods Preoperatively the range of motion (ROM) in the first MTP joint was determined, and the following questionnaires were used: Visual Analogue Scale (VAS) of pain, American Orthopedic Foot & Ankle Society (AOFAS) score, and Functional Foot Index (FFI). All of the patients experienced a significant restriction of the ROM in the first MTP joint. The VAS of pain median was 70, AOFAS 52, and FFI 5.8. Results AMIC was performed in 19 patients with first MTP joint OA. All the patients experienced a significant restriction of ROM in the first MTP joint. The VAS of pain median was 70, AOFAS 52, and FFI 5.8. By the third month, VAS of pain decreased to 25, FFI to 2.2; AOFAS median increased to 77, ROM to 60°. Six months after the surgery VAS of pain decreased to 10, FFI to 1.1; AOFAS median increased to 90, ROM to 65°. After a year of observation, VAS of pain decreased to 5, FFI to 1,0; AOFAS median increased to 92.5, ROM to 71.5°. Conclusion The results showed that a first MTP joint AMIC procedure can be a fairly effective method of surgical treatment that can relieve pain and significantly improve the quality of life of patients with first MTP joint OA.

Since the first case recorded in the end of 2019, the virus SARS-CоV-2 and the related lung disease COVID-19 have spread over the world and became a threat for public health. The drugs used for treatment of this disease (azithromycin and hydroxychloroquine) can prolong the QT interval on the electrocardiogram to increase the risk of pirouette tachycardia and sudden cardiac death. This article presents a review of potential risks related with the drug treatment of COVID-19 and provides recommendations for management of patients during the pandemic.

The aim: To investigate the effectiveness of complex treatment of purulent wounds with the use of high pressure aerodispersed mixture of antibacterial drug using sorbents and correction of hydrogen index.Materials and methods: In total, 54 patients were treated, out of which there were 26 patients with purulent wounds in the main group and 28 patients in the control group. Patients in the main group were treated according to the developed method, patients in the control group were treated with traditional antiseptics and water soluble ointments.Results: Within patients of the main group, there was a decrease in the duration of inflammatory process, decrease in the quantity of microorganisms in the wound; slight changes in the sensitivity of the microorganisms to antibacterial preparations, compared with the control group, which showed a high percentage of development of resistant strains; decreased edema and secretion from wounds, the early appearance of granulation.Conclusions: The use of the proposed approach reduces the microbial burden on the wound, accelerates its purification from purulent-necrotic tissues and reduces the duration of the inflammatory process due to the presence of the microbial factor.

Background Haematological malignancies harbouring rearrangements of the KMT2A gene represent a unique subtype of leukaemia, with biphenotypic clinical manifestations, a rapid and aggressive onset, and a generally poor prognosis. Chromosomal translocations involving KMT2A often cause the formation of oncogenic fusion genes, such as the most common translocation t(4;11)(q21;q23) producing the KMT2A‐AFF1 chimera. Aim The aim of this study was to confirm and review the cytogenetic and molecular features of the KMT2A‐rearranged RS4;11 cell line and put those in context with other reports of cell lines also harbouring a t(4;11) rearrangement. Methods and Results The main chromosomal rearrangements t(4;11)(q21;q23) and i(7q), described when the cell line was first established, were confirmed by fluorescence in situ hybridisation (FISH) and 24‐colour karyotyping by M‐FISH. Additional cytogenetic abnormalities were investigated by further FISH experiments, including the presence of trisomy 18 as a clonal abnormality and the discovery of one chromosome 8 being an i(8q), which indicates a duplication of the oncogene MYC. A homozygous deletion of 9p21 containing the tumour‐suppressor genes CDKN2A and CDKN2B was also revealed by FISH. The production of the fusion transcript KMT2A‐AFF1 arising from the der(11)t(4;11) was confirmed by RT‐PCR, but sequencing of the amplified fragment revealed the presence of multiple isoforms. Two transcript variants, resulting from alternative splicing, were identified differing in one glutamine residue in the translated protein. Conclusion As karyotype evolution is a common issue in cell lines, we highlight the need to monitor cell lines in order to re‐confirm their characteristics over time. We also reviewed the literature to provide a comparison of key features of several cell lines harbouring a t(4;11). This would guide scientists in selecting the most suitable research model for this particular type of KMT2A‐leukaemia.

Hydrophilic lung surfactant proteins have emerged as key immunomodulators aimed at recognition and clearance of pulmonary pathogens. Surfactant protein A (SP-A) is a surfactant-associated innate immune pattern recognition molecule, which is known to interact with a variety of pathogens, and display anti-microbial effects. SP-A, being carbohydrate pattern recognition molecule, has been suggested to have a wide range of innate immune functions against pathogens. In addition, SP-A can work against respiratory pathogens, including influenza A virus (IAV). Some pandemic pH1N1 strains resist neutralization by SP-A due to differences in the N-glycosylation of viral hemagglutinin (HA). Here, we provide evidence, for the first time, that a recombinant form of human SP-A (rfhSP-A) composed of α-helical neck and carbohydrate recognition domains can actually promote the IAV replication, as observed by an upregulation of M1 expression in lung epithelial cell line, A549, when challenged with pH1N1 and H3N2 IAV subtypes. rfhSP-A (10 μg/ml) bound neuraminidase (NA) (˜60 kDa), matrix protein 1 (M1) (˜25 kDa) and M2 (˜17 kDa) in a calcium dependent manner, as revealed by far western blotting, and direct binding ELISA. However, human full length native SP-A downregulated mRNA expression levels of M1 in A549 cells challenged with IAV subtypes. Furthermore, qPCR analysis showed that transcriptional levels of TNF-α, IL-12, IL-6, IFN-α and RANTES were enhanced following rfhSP-A treatment by both IAV subtypes at 6 h post-IAV infection of A549 lung epithelial cells. In the case of full length SP-A treatment, mRNA expression levels of TNF-α, and IL-6 were downregulated during the mid-to-late stage of IAV infection of A549 cells. Multiplex cytokine/chemokine array revealed enhanced levels of both IL-6 and TNF-α due to rfhSP-A treatment in the case of both IAV subtypes tested, while no significant effect was seen in the case of IL-12. Enhancement of IAV infection of pH1N1 and H3N2 subtypes by truncated rfhSP-A, concomitant with infection inhibition by full-length SP-A, appears to suggest that a complete SP-A molecule is required for protection against IAV. This is in contrast to a recombinant form of trimeric lectin domains of human SP-D (rfhSP-D) that acts as an entry inhibitor of IAV. KACST (14-MED258-20)

To investigate the effect of knee arthroscopic synovectomy (AS) on the disease activity, quality-of-life (QoL), and the functional status of patients with rheumatoid arthritis (RA). A retrospective analysis was conducted on the outcomes of AS performed on 138 RA patients; pre-surgery assessments were done using Disease Activity score (DAS 28) and Routine Assessment of Patient Index Data З (RAPID-3) on а multidimensional health-assessment questionnaire for disease activity, EuroQol-5D (EQ-5D) and the Short-Form Medical Outcomes Study (SF-36) for quality of life, and the Health Assessment Questionnaire (HAQ) for functional status. The pain response to SA was measured by а visual analogue score (VAS) and the Knee Society Score (KSS). All parameters assessed in the study showed significant positive changes: the activity of the disease decreased, and patients’ functional status and QoL improved. AS is effective treatment for recurrent synovitis of the knee in RA patients. This technique improves the functional status of patients and their quality of life and reduces the activity of the disease.

P53 protein is more frequently mutated in human tumours compared with the other proteins. While the majority of the p53 mutations, especially within its DNA-binding domain, lead to the loss of the wild-type function, there are accumulating data demonstrating that the p53 mutants gain tumour promoting activities; the latter triggers a revitalised interest in functional analysis of the p53 mutants. A systematic screening for p53 mutations in surgical materials from patients with glioma revealed a 378C>G mutation that creates a stop codon at the position of amino acid residue 126. The mutation eliminates the recognition site for the restriction endonuclease Sca I that allowed us to carry out RFLP analysis of DNA extracted from the clinical samples and suggests that this mutation is more frequent than is documented in the p53 databases. Both the ECV-304 and EJ cell lines, that probably originate from the bladder carcinoma T24 cell line, were confirmed to contain the homozygous 378C>G mutation but were shown to produce the p53 protein of expected full-length size detected by Western blotting. We provide evidence that the 378C>G mutation generates an alternative 3’ splice site (ss) which is more often used instead of the authentic upstream 3’ ss, driving the production of mRNA encoding the protein with the single amino acid deletion (p53ΔY126). Using endogenous expression, we demonstrated that the p53ΔY126 protein is nearly as active as the wild type protein in inducing the p21/Waf1 expression and apoptosis.

Mycobacterium tuberculosis can proficiently enter macrophages and diminish complement activation on its cell surface. Within macrophages, the mycobacterium can suppress macrophage apoptosis and survive within the intracellular environment. Previously, we have shown that complement regulatory proteins such as factor H may interfere with pathogen–macrophage interactions during tuberculosis infection. In this study, we show that Mycobacterium bovis BCG binds properdin, an upregulator of the complement alternative pathway. TSR4+5, a recombinant form of thrombospondin repeats 4 and 5 of human properdin expressed in tandem, which is an inhibitor of the alternative pathway, was also able to bind to M. bovis BCG. Properdin and TSR4+5 were found to inhibit uptake of M. bovis BCG by THP-1 macrophage cells in a dose-dependent manner. Quantitative real-time PCR revealed elevated pro-inflammatory responses (TNF-α, IL-1β, and IL-6) in the presence of properdin or TSR4+5, which gradually decreased over 6 h. Correspondingly, anti-inflammatory responses (IL-10 and TGF-β) showed suppressed levels of expression in the presence of properdin, which gradually increased over 6 h. Multiplex cytokine array analysis also revealed that properdin and TSR4+5 significantly enhanced the pro-inflammatory response (TNF-α, IL-1β, and IL-1α) at 24 h, which declined at 48 h, whereas the anti-inflammatory response (IL-10) was suppressed. Our results suggest that properdin may interfere with mycobacterial entry into macrophages via TSR4 and TSR5, particularly during the initial stages of infection, thus affecting the extracellular survival of the pathogen. This study offers novel insights into the non-complement related functions of properdin during host–pathogen interactions in tuberculosis.

In order to test the validity of the selected surgical technique as a way to manage persistent synovitis of the knee joint, as well as to slow down the cartilage and bone destruction, we studied the dynamics of biomarkers of inflammatory conditions, and bone and cartilage destruction after total arthroscopic synovectomy (TAS) of the knee joint. The sampling comprised 124 RA patients (158 knees) who had undergone the TAS procedure between 2003 and 2015. Before surgery the rheumatoid factor (IgM), C-reactive protein (CRP), erythrocyte sedimentation rate test was completed for all patients. Blood serum samples were collected (prior to surgery, and three, six and 12 months after surgery) and frozen at −70°С. The content of CRP, Matrix metalloproteinase-3 (MMP-3), Cartilage Oligomeric Matrix Protein, as well as cross-linked Ctelopeptides of types I and II were measured in blood serum by means of polarization fluoroimmunoassay with the use of a standard set of reagents. The average duration of the disease in the studied group was 8.7 ± 6.6 years. Concentration of the inflammation markers showed that only MMP-3 displayed statistical significance. The obtained results can be used as basis in assessing the efficiency and effectiveness of this method of treatment of persistent knee joint synovitis associated with the RA.

Microvolt T-wave alternans (MTA) is one of markers of electrical myocardial instability.To elucidate normal range of MTA parameters during bicycle exercise test (ET) in adolescent elite athletes.We examined 500 athletes aged 9-18 (mean 16.1 ± 2.2) years members of national teams of 27 kinds of sport. There were 333 females with mean age 16.0 ± 1.01 years and 167 males with mean age 16.3 ± 1.4. All athletes underwent ECG registration in clino- and ortho- positions and bicycle ET with automatic analysis of MTAT-wave alternans.Mean MTA value was 26 ± 13 (9-51) mV. MTA above 65 mV was registered in 10 persons (2.1%) 5 of whom had signs of overtraining. Sensitivity (Se) of MTA for detection of overtraining state was 21%, specificity (Sp)--99%, positive and negative predictive value--50 and 96%, respectively.Normal MTA range during ET is 9-51 mV. MTA values during ET do not depend on age and sex but are determined by duration of participation in sports and myocardial mass. MTA value is related to length of QT-interval. Increase of maximal MTA values above 65 mV is insensitive (Se 21%) but highly specific (Sp 99%) sign of overtraining in adolescent elite athletes.

Spliceosomes remove introns from primary gene transcripts. They assemble de novo on each intron through a series of steps that involve the incorporation of five snRNP particles and multiple non-snRNP proteins. In mammals, all the intermediate complexes have been characterized on one transcript (MINX), with the exception of the very first, complex E. We have purified this complex by two independent procedures using antibodies to either U1-A or PRPF40A proteins, which are known to associate at an early stage of assembly. We demonstrate that the purified complexes are functional in splicing using commitment assays. These complexes contain components expected to be in the E complex and a number of previously unrecognized factors, including survival of motor neurons (SMN) and proteins of the SMN-associated complex. Depletion of the SMN complex proteins from nuclear extracts inhibits formation of the E complex and causes non-productive complexes to accumulate. This suggests that the SMN complex stabilizes the association of U1 and U2 snRNPs with pre-mRNA. In addition, the antibody to PRPF40A precipitated U2 snRNPs from nuclear extracts, indicating that PRPF40A associates with U2 snRNPs.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a severe premature aging syndrome that affects children. These children display characteristics associated with normal aging and die young usually from cardiovascular problems or stroke. Classical HGPS is caused by mutations in the gene encoding the nuclear structural protein lamin A. This mutation leads to a novel version of lamin A that retains a farnesyl group from its processing. This protein is called Progerin and is toxic to cellular function. Pre-lamin A is an immature version of lamin A and also has a farnesylation modification, which is cleaved in the maturation process to create lamin A.

Primary gene transcripts of eukaryotes contain introns, which are removed during processing by splicing machinery. Biochemical studies In vitro have identified a specific pathway in which introns are recognised and spliced out. This occurs by progressive formation of spliceosomal complexes designated as E, A, B, and C. The composition and structure of these spliceosomal conformations have been characterised in many detail. In contrast, transitions between the complexes and the intermediates of these reactions are currently less clear. We have previously isolated a novel 35S U5 snRNP from HeLa nuclear extracts. The protein composition of this particle differed from the canonical 20S U5 snRNPs but was remarkably similar to the activated B* spliceosomes. Based on this observation we have proposed a hypothesis that 35S U5 snRNPs represent a dissociation product of the spliceosome after both transesterification reactions are completed. Here we provide experimental evidence that 35S U5 snRNPs are generated from the activated B* spliceosomes during In vitro splicing.

Major structural changes occur in the spliceosome during its transition from the fully assembled complex B to the catalytically activated spliceosome. To understand the rearrangement, it is necessary to know the detailed three-dimensional structures of these complexes. Here, we have immunoaffinity-purified human spliceosomes (designated B Delta U1) at a stage after U4/U6.U5 tri-snRNP integration but before activation, and have determined the three-dimensional structure of B Delta U1 by single-particle electron cryomicroscopy at a resolution of approximately 40 A. The overall size of the complex is about 370 x 270 x 170 A. The three-dimensional structure features a roughly triangular body linked to a head domain in variable orientations. The body is very similar in size and shape to the isolated U4/U6.U5 tri-snRNP. This provides initial insight into the structural organization of complex B.

Major structural changes occur in the spliceosome during its activation just before catalyzing the splicing of pre–messenger RNAs (pre-mRNAs). Whereas changes in small nuclear RNA (snRNA) conformation are well documented, little is known about remodeling of small nuclear ribonucleoprotein (snRNP) structures during spliceosome activation. Here, human 45 S activated spliceosomes and a previously unknown 35 S U5 snRNP were isolated by immunoaffinity selection and were characterized by mass spectrometry. Comparison of their protein components with those of other snRNP and spliceosomal complexes revealed a major change in protein composition during spliceosome activation. Our data also suggest that the U5 snRNP is dramatically remodeled at this stage, with the Prp19 complex and other factors tightly associating, possibly in exchange for other U5 proteins, and suggest that after catalysis the remodeled U5 is eventually released from the postsplicing complex as a 35 S snRNP particle.

The RBMY gene family is found on the Y chromosome of all mammals, and microdeletions are strongly associated with infertility in men. RBMY expresses RBM only in the nuclei of germ cells, whereas its X chromosome homologue, RBMX, expresses hnRNP G ubiquitously. We show here that RBM, hnRNP G and a novel testis-specific relative, termed hnRNP G-T, interact with Tra2beta, an activator of pre-mRNA splicing that is ubiquitous but highly expressed in testis. Endogenous hnRNP G and Tra2beta proteins are associated in HeLa nuclear extracts. RBM and Tra2beta co-localize in two major domains in human spermatocyte nuclei. Phosphorylation enhanced the interaction and reduced competing RNA binding to the interaction domains. Incubation with the protein interaction domain of RBM inhibited splicing in vitro of a specific pre-mRNA substrate containing an essential enhancer bound by Tra2beta. The RNA-binding domain of RBM affected 5' splice site selection. We conclude that the hnRNP G family of proteins is involved in pre-mRNA splicing and infer that RBM may be involved in Tra2beta-dependent splicing in spermatocytes.

A case of a rare disease - Andersen-Tawil the syndrome (ATS) is presented. Diagnosis of ATS, 7-th molecular-genetic variant of long QT syndrome was made basing on the characteristic clinical picture (periodic stress induced syncopal states), data of ECG and its Holter monitoring (pronounced QT prolongation, bouts of polymorphic bidirectional ventricular tachycardia), typical dysmorphic features (low-set ears, small mandible, brachydactyly, fifth-digit clinodactyly). However mutation of the KCNJ2 gene typical for this variant was not detected. Problems of pathogenesis, diagnostics, and treatment of the disease are discussed with special stress on class IC antiarrhythmic drugs.

Sudden cardiac death (SCD) of two sisters from a family with combined progressive impairment of cardiac conduction system (CCS) is described. These two sisters (hetero-ovular twins) died suddenly in a kindergarten at the background of physical and emotional effort in the age of 4.8 and 5 years. Resuscitation and electrical defibrillation conducted in one of them after 20 min of heart arrest were not effective. Autopsy gave no clear-cut data relative to pathology of the heart and other organs. Analysis of preserved ECGs showed that in the age of 8 months both sisters had sharp leftward/upward deviation of electric axis of the heart (-41 in one sister and -43 in another). In 2 years these deviations became even more pronounced reaching -63. This was combined with small q-waves and tall R waves in leads I and aVL what corresponded to signs of block of the anterior branch of bundle of His and could be a manifestation of hereditary disease with progressive CCS involvement first of all of the Lev-Lenegre disease.

Aim of the study was determination of physiological limits of QT-intervals and its derivative values in healthy children and adolescents during graded exercise tests. We examined 100 healthy boys and girls aged 11-15 years (mean age 13.4+/-2.1 years) and performed electrocardiography at rest and standard veloergometry (VEM) in all of them. We analyzed corrected intervals according to Bazett (QTc=QT/RR) and Fredericia (FQTc/3RR) formulas. Hysteresis QTc was calculated as difference between QTc durations during recovery and exercise at same heart rate (HR) Baseline HR before VEM exceeded rhythm on resting electrocardiogram by 5-15 bpm (84+/-8 vs 70+/-6, respectively, plt;0.05) Increase of HR at exercise (mean 172+/-11 bpm) was similar in both sexes. QT interval decreased by 7-10% (18-31 ms) per each 25 w (plt;0.05). Values obtained at determination of FQTc we found values 26-52 ms lower than those calculated by the Bazett formula in the process of whole test. Determination of FQTc compared with calculation by Bazett formula revealed more pronounced (10% from baseline level) shortening of FQT at peak exercise. QT calculated by the Bazett formula at 100 w did not differ from baseline level with tendency to higher level. Corrected QT according to the most often used Bazett formula was maximal at the first stage of exercise (25 w) and did not exceed 450 ms in boys and 460ms in girls. Maximal QTc lengthening in the process of test did not exceed 50 ms in any of the examined persons. Hysteresis of QTc interval was equal to 21+/-6 (15-25) ms. The conclusion was made that algorithm of assessment of QT interval changes during exercise test should include initial values of QTc calculated according to the Bazett formula, maximal QTc value, level of exercise at which it was registered, maximal increase of QTc during exercise, and QTc interval hysteresis.

There is little quantitative information regarding how much splicing occurs co-transcriptionally in higher eukaryotes, and it remains unclear where precisely splicing occurs in the nucleus. Here we determine the global extent of co- and post-transcriptional splicing in mammalian cells, and their respective subnuclear locations, using antibodies that specifically recognize phosphorylated SF3b155 (P-SF3b155) found only in catalytically activated/active spliceosomes. Quantification of chromatin- and nucleoplasm-associated P-SF3b155 after fractionation of HeLa cell nuclei, reveals that ~80% of pre-mRNA splicing occurs co-transcriptionally. Active spliceosomes localize in situ to regions of decompacted chromatin, at the periphery of or within nuclear speckles. Immunofluorescence microscopy with anti-P-SF3b155 antibodies, coupled with transcription inhibition and a block in splicing after SF3b155 phosphorylation, indicates that post-transcriptional splicing occurs in nuclear speckles and that release of post-transcriptionally spliced mRNA from speckles is coupled to the nuclear mRNA export pathway. Our data provide new insights into when and where splicing occurs in cells.

Background: Radiotherapy-induced DNA double-strand breaks (DSBs) are critical cytotoxic lesions. Inherited defects in DNA DSB repair pathways lead to hypersensitivity to ionising radiation, immunodeficiency and increased cancer incidence. A patient with xeroderma pigmentosum complementation group C, with a scalp angiosarcoma, exhibited dramatic clinical radiosensitivity following radiotherapy, resulting in death. A fibroblast cell line from non-affected skin (XP14BRneo17) was hypersensitive to ionising radiation and defective in DNA DSB repair. Aim: To determine the genetic defect causing cellular radiation hypersensitivity in XP14BRneo17 cells. Methods: Functional genetic complementation whereby copies of human chromosomes containing genes involved in DNA DSB repair (chromosomes 2, 5, 8 10, 13 and 22) were individually transferred to XP14BRneo17 cells in an attempt to correct the radiation hypersensitivity. Clonogenic survival assays and g-H2AX immunofluorescence were conducted to measure radiation sensitivity and repair of DNA DSBs. DNA sequencing of defective DNA repair genes was performed. Results: Transfer of chromosome 8 (location of DNAPKcs gene) and transfection of a mammalian expression construct containing the DNA-PKcs cDNA restored normal ionising radiation sensitivity and repair of DNA DSBs in XP14BRneo17 cells. DNA sequencing of the DNA-PKcs coding region revealed a 249-bp deletion (between base pairs 3656 and 3904) encompassing exon 31 of the gene. Conclusion: We provide evidence of a novel splice variant of the DNA-PKcs gene associated with radiosensitivity in a patient with xeroderma pigmentosum and report the first double mutant in distinct DNA repair pathways being consistent with viability.

Aim of this study was determination of incidence and structure of sudden out of hospital cardiac death (SCD) in subjects aged 1-45 years in Moscow. We analyzed rate and structure of SD among persons who had succumbed in 2005-2007 in several districts of Moscow with population of 2,502,836. Of the total number of 19,557 autopsies 7702 (39.4% or 92/100 000/year) and 1265 (6.5%, 19.4% of autopsies in this age group, or 16.8/100000/year) in all age groups and in the age 1-45 years, respectively, were performed because of SCD. In most cases (44%) age at SCD was 41-45 years, only in 1% of cases it was less than 18 years. Eighty two percent of autopsied SD victims were men. Most frequent postmortem diagnosis was "cardiomyopathy" (69% overall, 80-96% in the age 19-35 years) established on the basis of detection of both specific and nonspecific changes in the myocardium. In 25% of SD cases in the age group before 18 years congenital heart disease was found. After 35 years rose portion of diseases of ischemic nature (22-32%). Percentages of hypertensive disease and other diseases among all diagnoses were 7 and 1, respectively. Beginning with 19 years in large percentage of cases presence of alcohol in blood was detected. This percentage was especially high in the age group 19-25 years (66.6%). SCD of most persons aged 20-45 years was registered at home, while in 50% of younger persons it occurred outside home often during physical effort. Rate of SCD, its circumstances and structure elicited in this study might serve as basis for elaboration of the system of early detection of groups of risk and prevention of SD in young age.

Aim of the study was to determine dynamics of main parameters of heart rhythm in children during first days of life. Material and methods. We examined 30 healthy newborns (14 boys and 16 girls) aged 1-4 days (2,6+/-1,3 days). Criteria of inclusion were healthy mother, normal course of pregnancy and delivery, gestational age at birth 38-40 weeks, body mass at birth 3200-3600 g; Algar score 8/9, no cardiovascular abnormalities at physical examination, informed consent of parents. On days 1-2 and 4 of life we carried out 12 lead ECG at rest and 24-hour Holter monitoring with assessment of heart rate (HR), arrhythmias, dynamics of QT-interval, HR variability (HRV) using methods "Time Domain" and "deceleration (DC)/acceleration capacity (AC)". Results. Average 24-hour HR was 138+/-17 beats/min, maximal pauses of sinus rhythm - 990 ms. Supraventricular extrasystoles (10/24 hours) were registered in 35%, ventricular extrasystoles (5/24 hours) in 15% of children. Values of HRV were as follows: N 442 (408-72) ms; SDNN 52 (43-75) ms; SDNNi 36 (35-61) ms; SDNNi24 (22-37) ms; SSD 14 (10-20) ms; NN50% 0,98(0-2,4)%. Analysis of D/ showed the following values: D = +3.43 +/- 0.54 (2.16-4.14),=-3.65+/-0.65 (from - 2.13 to - 4.38). Analysis of interval QT values: average 24-hour QT (QTo) - 293+/-10 (278-308) ms, maximal - 342+/-38 (328-360) ms; mean 24-hour QT 442+/-14 (428-470) ms; QT 204+/-11 (191-220) ms; QT - 308+/-17(284-337) ms, sl Q/RR 0.37 (0.304-0.505); intercept QT/RR 126 (92.0-151.0), correlation coefficient () QT/RR 0.65 (0.56-0.84). On day 4 compared with day 1 there were significantly higher values of correlation (r) QT/RR and lower values of sl QT/RR. Conclusions. During first days of life children can have supraventricular and ventricular extrasystoles (not more than 10 per 24 hours). First 24 hours of life are characterized by most pronounced signs of myocardial instability according to data of assessment of QT dynamics. HRV parameters of healthy newborns are characterized by high level of sympathetic activity, symmetry of deceleration capacity/acceleration capacity parameters.

Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post-translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B'. Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.

PURPOSE. Pre-mRNA processing factor 31 (PRPF31) is a ubiquitous protein needed for the assembly of the pre-mRNA splicing machinery. It has been shown that mutations in this gene cause autosomal dominant retinitis pigmentosa 11 (RP11), which is characterized by rod-cell degeneration. Interestingly, mutations in this ubiquitously expressed gene do not lead to phenotypes other than retinal malfunction. Furthermore, the dominant inheritance pattern has shown incomplete penetrance, which poses interesting questions about the disease mechanism of RP11. METHODS. To characterize PRPF31 function in the rod cells, two animal models have been generated. One was a heterozygous knock-in mouse (Prpf31(A216P/+)) carrying a point mutation p.A216P, which has previously been identified in RP11 patients. The second was a heterozygous knockout mouse (Prpf31(+/-)). Retinal degeneration in RP11 mouse models was monitored by electroretinography and histology. RESULTS. Generation of the mouse models is presented, as are results of ERGs and retinal morphology. No degenerative phenotype on fundus examination was found in Prpf31(A216P/+) and Prpf31(+/-) mice. Prpf31(A216P/A216P) and Prpf31(-/-) genotypes were embryonic lethal. CONCLUSIONS. The results imply that Prpf31 is necessary for survival, and there is no compensation mechanism in mouse for the lack of this splicing factor. The authors suggest that p.A216P mutation in Prpf31 does not exert a dominant negative effect and that one Prpf31 wild-type allele is sufficient for maintenance of the healthy retina in mice. (Less)

Aim of the investigation consisted in to study parameters of " QT-dynamics " in healthy children and adolescents. We examined 26 children aged 7 - 17 years (mean 11,6 +/- 7 years) - 7 girls (9,3 +/- 2,3 years) and 19 boys (12,5 +/- 3,6 years). Holter monitoring with manual and automatic estimation of QT and QTc intervals. Dynamics of magnitude of QT interval was determined with the use of equation of linear regression Y = aX+b, where aX - coefficient of linear regression QT/RR (slope QT/RR), b - shear coefficient (intercept QT/RR). Values of QT interval at automatic analysis were 368,8 +/- 18,04 (340 - 410) ms for absolute QT and 424 +/- 14,99 (390 - 450) ms - for QTc, 289,4 +/- 14,6 (260 - 320). Mean values of " QT dynamics " were for slope QT/RR: 0,18 +/- 0,03 (0,13 - 0,24) for 24 hours, 0,16 +/- 0,03 (0,10 - 0,22) for daytime period, and 0,11 +/- 0,13 (0,05 - 0,18) for night time period; for intercept: QT/RR - 230,4 +/- 20,7 (192 - 282) for 24 hours, 299 +/- 25,5 (258 - 364) for day time period, and 242,7 +/- 21,8 (204 - 295) for night time period. Coefficient of correlation of intervals QT and RR (r QT/RR) was 0,79 +/- 0,05 (0,69 - 0,89) for 24 hours; 0,68 +/- 0,07 (0,52 - 0,82) for day time period, and 0,52 +/- 0,13 (0,19 - 0,74) for night time period. All values of " QT-dynamics " significantly differed from each other during various periods of day (p0,05). Negative relation was noted between 24 hour slope and intercept QT/RR (r= -0,88, p0,05), diurnal (r= -0,87, p0,05) and nocturnal slope and intercept QT/RR (r= -0,72, p0,05), positive relation was noted between 24 hour values of slope QT/RR and QT (r=0,49, p0,05), and intercept QT/RR with absolute QT (r=0,41, p0,05). Significantly higher values (p0,01) of slope QT/RR were in girls. Thus, increase of slope QT/RR is noted during augmentation of tone of sympathetic part of vegetative nervous system, lowering - when vagal influences are also enhanced. Intercept QT/RR appears to be combination parameter, increase of which reflects both lengthening of absolute mean value of QT interval and reciprocal nocturnal lowering of the slope parameter. It is necessary to assesses clinical and diagnostic significance of changes of novel parameters of " QT-dynamics " on the basis of normative sexual-aging values and physiological mechanisms, determining their variability.

Aspirin, dipyridamole, cilostazol, thienopyridines and glycoprotein IIb/IIIa inhibitors represent the classical examples of the established antiplatelet agents commonly used for the secondary prevention in patients after vascular events. Obviously, the era of expanding antiplatelet regimens and indications may require new agents as the substitutes, or additions to the available strategies. However, recent results of the majority of antiplatelet trials strongly suggest boarder line advantages in clinical outcomes, and higher associated bleeding risks with the novel antiplatelet agents or/and regimens. Moreover, unexpected failures, such as lack of efficacy of clopidogrel and aspirin combination for ischaemic stroke prevention (MATCH), or use of the same antiplatelet regimen for the primary vascular prevention (CHARISMA) raise legitimate concerns that the concept 'the more the better' may not be valid. Broad use of statins, angiotensin receptor blockers and selective serotonin reuptake inhibitors may be in part responsible for the lack of impressive results with the antiplatelet therapy because each of these drug classes per se inhibits platelets. In this review, we discuss the available evidence and potential clinical significance of these findings.

In eukaryotes, pre-mRNA exons are interrupted by large noncoding introns. Alternative selection of exons and nucleotide-exact removal of introns are performed by the spliceosome, a highly dynamic macromolecular machine. U4/U6.U5 tri-snRNP is the largest and most conserved building block of the spliceosome. By 3D electron cryomicroscopy and labeling, the exon-aligning U5 snRNA loop I is localized at the center of the tetrahedrally shaped tri-snRNP reconstructed to approximately 2.1 nm resolution in vitrified ice. Independent 3D reconstructions of its subunits, U4/U6 and U5 snRNPs, show how U4/U6 and U5 combine to form tri-snRNP and, together with labeling experiments, indicate a close proximity of the spliceosomal core components U5 snRNA loop I and U4/U6 at the center of tri-snRNP. We suggest that this central tri-snRNP region may be the site to which the prespliceosomal U2 snRNA has to approach closely during formation of the catalytic core of the spliceosome.

Aim. To determine limits of interval QT in children and adolescents aged 0-17 years and to detect possible ethnic differences of its changes. Material and Method. Twelve lead ECGs were recorded in 1531 subjects without overt cardiovascular pathology (47.2% females, 52.8 males, 57.3% of Slavic and 42.7% of Buryat i.e. mongoloid ethnicity). Results. Corrected QT interval (QTc) exceeded 440, 460 and 480 ms in 2.3, 1.05, and 0.26% of children. Prolongation of QTc was found in 0.46% and 0.11% of Slavic and Buryat children, respectively (p=ns). Starting from the age of 8 years girls had longer QTc than boys (454.1+/-15.2 and 438.3+/-8.4 ms, respectively, p0.05). QTc less than 350 ms was registered in 12 children (0.78%). Eight of these children with mean QTc 329.1+/-32.3 ms had family history of syncope or sudden death. Variability of absolute QT values was 8+/-14.3 ms (maximally up to 40 ms). Conclusions. QT interval is prolonged when QTc duration exceeds 440 ms in children younger than 8 years and in male adolescents or 460 ms in girls aged 8 years or older and in children during first year of life. QT interval is shortened when QTc is less than 350 ms (1st degree of shortening). In children with QTc below 330 ms (2nd degree of shortening) short QT syndrome should be excluded. Normal variability of absolute QT value during sinus arrhythm on ECG at rest does not exceed 40 ms.

To assess parameters of heart rate turbulence in patients with ventricular extrasystoles (VES).Patients with VES during Holter ECG monitoring (n=65, age 1-17 years, 32 girls, 33 boys) including 3 with long QT syndrome, 4 with catecholaminergic ventricular tachycardia, 1 with arrhythmogenic right ventricular dysplasia, 4 with dilated cardiomyopathy, 2 with Brugada syndrome, 3 with Duchenne myopathy, and 48 with idiopathic VES. Parameters HRT onset and slope were calculated for 1678 VES. During follow-up (6+/-4.9 years) deaths, sudden deaths, and such complications as syncope, cardiac dilatation, heart failure were registered in 26 patients.HRT slope exceeded 2.5 ms/RR in all patients. HRT onset was0 in patients with catecholaminergic ventricular tachycardia, dilated cardiomyopathy, and Duchenne myopathy. The following complications occurred in 7 of 8 patients (87.5%) with HRT onset0: syncope, sudden deaths (2 patients with dilated cardiomyopathy), dilatation of cardiac chambers, tolerance to antiarrhythmic therapy. Patients with long QT syndrome and arrhythmogenic right ventricular dysplasia (including 2 who afterwards died suddenly) had normal HRT onset. The sensitivity and specificity of HRT onset measurement for unfavorable prognosis of VES was 27 and 97%, respectively. No relationship was found between HRT slope and onset (r=-0.057).Pathological HRT (mainly represented by changes of HRT onset) are characteristic for organic myocardial diseases. Therefore detection of abnormal HRT dictates necessity of exclusion of organic cardiac pathology.

The U5 snRNP plays an essential role in both U2- and U12-dependent splicing. Here, we have characterized a 52-kDa protein associated with the human U5 snRNP, designated U5-52K. Protein sequencing revealed that U5-52K is identical to the CD2BP2, which interacts with the cytoplasmic portion of the human T-cell surface protein CD2. Consistent with it associating with an snRNP, immunofluorescence studies demonstrated that the 52K protein is predominantly located in the nucleoplasm of HeLa cells, where it overlaps, at least in part, with splicing-factor compartments (or “speckles”). We further demonstrate that the 52K protein is a constituent of the 20S U5 snRNP, but is not found in U4/U6.U5 tri-snRNPs. Thus, it is the only 20S U5-specific protein that is not integrated into the tri-snRNP and resembles, in this respect, the U4/U6 di-snRNP assembly factor Prp24p/p110. Yeast two-hybrid screening and pulldown assays revealed that the 52K protein interacts with the U5-specific 102K and 15K proteins, suggesting that these interactions are responsible for its integration into the U5 particle. The N-terminal two-thirds of 52K interact with the 102K protein, whereas its C-terminal GYF-domain binds the 15K protein. As the latter lacks a proline-rich tract, our data indicate that a GYF-domain can also engage in specific protein–protein interactions in a polyproline-independent manner. Interestingly, the U5-102K protein has been shown previously to play an essential role in tri-snRNP formation, binding the U4/U6-61K protein. The interaction of 52K with a tri-snRNP bridging protein, coupled with its absence from the tri-snRNP, suggests it might function in tri-snRNP assembly.

Right ventricular arrhythmogenic dysplasia (RVAD) is a state with high risk of sudden death in young patients. Early diagnosis of RVAD can facilitate sudden death prevention.To assess prevalence of electrocardiographical signs of RVAD among children with "idiopathic" tachyarrhythmias.Patients without organic heart disease or overt noncardiac causes of arrhythmia aged 4-17 years (n=134, mean age 12+/-4.5 years, 56 girls and 76 boys) including 82 patients with5000 extrasystoles VE 24 hours and 52 patients with ventricular tachycardia (VT). All patients had QTc interval440 ms.Twelve lead ECGs from all patients were analyzed for determination of morphology of arrhythmia and presence of "major" (epsilon wave and QRS duration110 ms in V(1)-V(3)) and "minor" (VT with left bundle brunch block - LBBB, VE1000/24 hours, T-wave inversion in V(2) and V(3)) diagnostic criteria for RVAD (W.McKenna, 1994; D.Corrado, 2000).ECG signs of RVAD were found in 28 of 58 (48.3%) of patients with VE and in 1 of 24 patients (4.2%) with atrial extrasystoles. Among patients with VE 27 (96.4%) had extrasystoles with LBBB morphology and 1 (3.6%) - with right bundle brunch block (RBBB) morphology. Combination of major and minor criteria sufficient for diagnosis of RVAD was found in 19% (8 of 42) of patients with VE with LBBB. Among 52 patients with VT 21 had polymorphic VT and 31 - monomorphic VT (16 with LBBB and 15 with RBBB). Epsilon wave was present in 56.3% (9/15) of patients with monomorphic VT and LBBB, in 4.8% (1/21) of patients with polymorphic VT and in none of the patients with monomorphic VT and RBBB. QRS duration exceeded 110 ms in 2 (12.5%), 2 (9.5%) and 0 patients among those with monomorphic VT and LBBB, polymorphic VT, and monomorphic VT and RBBB, respectively. Among patients with monomorphic VT and LBBB 37.5% (6/16) had combination of ECG criteria sufficient for diagnosis of RVAD. In 3 patients epsilon wave was registered in lead V(1) immediately prior to VT and disappeared after VT cessation. Implications. It can be suggested that presence of polymorphic VT or VE with LBBB morphology and specific ECG changes (first of all epsilon wave and QRS widening) distinguishes a group of patients with high risk of RVAD which requires thorough cardiological examination and dynamic observation.

During catalytic activation of the spliceosome, snRNP remodeling events occur, leading to the formation of a 35S U5 snRNP that contains a large group of proteins, including Prp19 and CDC5, not found in 20S U5 snRNPs. To investigate the function of 35S U5 proteins, we immunoaffinity purified human spliceosomes that had not yet undergone catalytic activation (designated BDeltaU1), which contained U2, U4, U5, and U6, but lacked U1 snRNA. Comparison of the protein compositions of BDeltaU1 and activated B* spliceosomes revealed that, whereas U4/U6 snRNP proteins are stably associated with BDeltaU1 spliceosomes, 35S U5-associated proteins (which are present in B*) are largely absent, suggesting that they are dispensable for complex B formation. Indeed, immunodepletion/complementation experiments demonstrated that a subset of 35S U5 proteins including Prp19, which form a stable heteromeric complex, are required prior to catalytic step 1 of splicing, but not for stable integration of U4/U6.U5 tri-snRNPs. Thus, comparison of the proteomes of spliceosomal complexes at defined stages can provide information as to which proteins function as a group at a particular step of splicing.

Long QT-interval is one of most important predictors of risk of development of life threatening arrhythmias and sudden death. Correct measurement of QT-interval is essential for diagnosis of its prolongation. At present the Bazett formula for calculation of corrected QT (QT(c) = QT/ radical (RR)) is a standard method of QT estimation. However historically in Russia calculation of predicted QT (QT(k) = k radical (RR)) QT(k) has become an accepted technique. Furthermore many authors in this country apply criteria created for QT(c) for interpretation of QT(k) values. This results in hyperdiagnosis of QT prolongation in unaffected persons, erroneous conclusions on harmless nature of this condition, and underestimation of risk in patients with real long QT syndrome. Thus it is vital to proclaim calculation of QT(c) an obligatory standard and to use existing international criteria for its interpretation.

To assess frequency of short QT interval among members of families with history of young age sudden death.Eleven children aged 3-16 years (mean age 11.2+/-4.2 years) from families with cases of sudden death at young age (40 years) not caused by an obvious disease according to autopsy data. None of the probands had overt heart disease, life threatening arrhythmias, long QT syndrome.Predicted QT value was estimated according to the P. Rautaharju formula [QT(p)=656/(1+HR/100)], and values equal to 80 and 88% of QT(p) (QT(p)80 and QT(p)88) were determined. Corrected QT interval duration (QT(c)) was calculated for all these values with the use of the Bazett formula.Values of corrected QT(c)88 370 ms and 350 ms were considered minimal for heart rates above and below 78 bpm, respectively. QT shortening to the level of QT(p)80 at heart rates above 70 bpm corresponded to QT(c) values330 ms. Nine of 11 children (81.1%) had QT interval equal to or less than QT(p)88. Three probands with attacks of syncope had QT equal to QT(p)80, high concentration of sudden deaths in family (up to 30% of family members), and high prevalence of shortened QT80% among family members.Shortened QT interval is characteristic for children with family history of sudden death at young age. Registration of QT duration equal to or shorter than 88% of predicted value requires exclusion of diseases and syndromes with increased risk of sudden death.

To elucidate clinical and electrocardiographical characteristics of children and adolescents with malignant idiopathic polymorphic ventricular tachycardia.Patients aged 6-14 years (n=16) with polymorphic bidirectional tachycardia registered either on standard 12-lead ECG (n=6), or during treadmill exercise test (n=2) and Holter ECG monitoring (n=8). None of the patients had overt heart or coronary artery disease, chronic extracardiac pathology or electrolyte disturbances.There was no QT, QT(s) prolongations, or ST segment elevations in right precordial leads. Eleven patients (68.8%) had shortening of PR interval -110 ms without signs of Wolf-Parkinson-White syndrome. All patients with shortened PR had history of syncopal states (9.1+/-9.2 per year), 4 patients (36.4%) had family history of sudden death in young age. Among patients without PR shortening 2 (40%) had history of syncopal attacks (1-2 or 0.6+/-0.89 per year). All patients with short PR had bradycardia while in sinus rhythm (55.5+/-9.1 bpm compared with 78.1+/-2.8 bpm in patients without PR shortening).Shortened PR and bradycardia were found to be associated with frequent attacks of syncope in this series of children with polymorphic tachycardia. Therefore combination of short PR, bradycardia and polymorphic ventricular tachycardia was suggested to comprise clinico-electrocardiographic syndrome with high risk of malignant ventricular tachyarrhythmia and sudden death.

A growing body of evidence supports the coordination of pre- mRNA processing and transcriptional regulation. We demonstrate here that mammalian PRP4 kinase (PRP4K) is associated with complexes involved in both of these processes. PRP4K is implicated in pre-mRNA splicing as the homologue of the Schizosaccharomyces pombe pre-mRNA splicing kinase Prp4p, and it is enriched in SC35-containing nuclear splicing speckles. RNA interference of Caenorhabditis elegans PRP4K indicates that it is essential in metazoans. In support of a role for PRP4K in pre-mRNA splicing, we identified PRP6, SWAP, and pinin as interacting proteins and demonstrated that PRP4K is a U5 snRNP- associated kinase. In addition, BRG1 and N-CoR, components of nuclear hormone coactivator and corepressor complexes, also interact with PRP4K. PRP4K coimmunoprecipitates with N-CoR, BRG1, pinin, and PRP6, and we present data suggesting that PRP6 and BRG1 are substrates of this kinase. Lastly, PRP4K, BRG1, and PRP6 can be purified as components of the N-CoR-2 complex, and affinity-purified PRP4K/N-CoR complexes exhibit deacetylase activity. We suggest that PRP4K is an essential kinase that, in association with the both U5 snRNP and N-CoR deacetylase complexes, demonstrates a possible coordination of pre-mRNA splicing with chromatin remodeling events involved in transcriptional regulation.

In each round of nuclear pre‐mRNA splicing, the U4/U6·U5 tri‐snRNP must be assembled from U4/U6 and U5 snRNPs, a reaction that is at present poorly understood. We have characterized a 61 kDa protein (61K) found in human U4/U6·U5 tri‐snRNPs, which is homologous to yeast Prp31p, and show that it is required for this step. Immunodepletion of protein 61K from HeLa nuclear extracts inhibits tri‐snRNP formation and subsequent spliceosome assembly and pre‐mRNA splicing. Significantly, complementation with recombinant 61K protein restores each of these steps. Protein 61K is operationally defined as U4/U6 snRNP‐specific as it remains bound to this particle at salt concentrations where the tri‐snRNP dissociates. However, as shown by two‐hybrid analysis and biochemical assays, protein 61K also interacts specifically with the U5 snRNP‐associated 102K protein, indicating that it physically tethers U4/U6 to the U5 snRNP to yield the tri‐snRNP. Interestingly, protein 61K is encoded by a gene ( PRPF31 ) that has been shown to be linked to autosomal dominant retinitis pigmentosa. Thus, our studies suggest that disruptions in tri‐snRNP formation and function resulting from mutations in the 61K protein may contribute to the manifestation of this disease.