Assembly defects of human tRNA splicing endonuclease contribute to impaired pre-tRNA processing in pontocerebellar hypoplasia

Mutations within subunits of the tRNA splicing endonuclease complex (TSEN) are associated with pontocerebellar hypoplasia (PCH). Here the authors show that tRNA intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers, and provide evidence that modulation of TSEN stability may contribute to PCH phenotypes.Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-angstrom resolution X-ray crystal structure of a TSEN15-34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.Genetics of disease, diagnosis and treatmen

Measurement of the Atmospheric Muon Spectrum from 20 to 3000 GeV

The absolute muon flux between 20 GeV and 3000 GeV is measured with the L3 magnetic muon spectrometer for zenith angles ranging from 0 degree to 58 degree. Due to the large exposure of about 150 m2 sr d, and the excellent momentum resolution of the L3 muon chambers, a precision of 2.3 % at 150 GeV in the vertical direction is achieved. The ratio of positive to negative muons is studied between 20 GeV and 500 GeV, and the average vertical muon charge ratio is found to be 1.285 +- 0.003 (stat.) +- 0.019 (syst.).Comment: Total 32 pages, 9Figure

Characterization of Human DNA Polymerase Delta and Its Subassemblies Reconstituted by Expression in the Multibac System

Mammalian DNA polymerase δ (Pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and DNA repair processes. We have reconstituted human Pol δ complexes in insect cells infected with a single baculovirus into which one or more subunits were assembled. This system allowed for the efficient expression of the tetrameric Pol δ holoenzyme, the p125/p50 core dimer, the core+p68 trimer and the core+p12 trimer, as well as the p125 catalytic subunit. These were isolated in milligram amounts with reproducible purity and specific activities by a highly standardized protocol. We have systematically compared their activities in order to gain insights into the roles of the p12 and p68 subunits, as well as their responses to PCNA. The relative specific activities (apparent kcat) of the Pol δ holoenzyme, core+p68, core+p12 and p125/p50 core were 100, 109, 40, and 29. The corresponding apparent Kd's for PCNA were 7.1, 8.7, 9.3 and 73 nM. Our results support the hypothesis that Pol δ interacts with PCNA through multiple interactions, and that there may be a redundancy in binding interactions that may permit Pol δ to adopt flexible configurations with PCNA. The abilities of the Pol δ complexes to fully extend singly primed M13 DNA were examined. All the subassemblies except the core+p68 were defective in their abilities to completely extend the primer, showing that the p68 subunit has an important function in synthesis of long stretches of DNA in this assay. The core+p68 trimer could be reconstituted by addition of p12

Внедрение и принцип работы системы сейсмического мониторинга горного массива для работы в условиях ООО "Шахта "Усковская"

The status of the Silicon Microvertex Detector (SMD) and its installation into the LEP-L3 experiment are presented, highlighting novel features and sophisticated techniques. Preliminary results based on 1993 data are given and compared with Monte Carlo predictions, to understand the detector performances and its tracking capabilities