The intrinsically disordered TSSC4 protein acts as a helicase inhibitor, placeholder and multi-interaction coordinator during snRNP assembly and recycling

Biogenesis of spliceosomal small nuclear ribonucleoproteins (snRNPs) and their recycling after splicing require numerous assembly/recycling factors whose modes of action are often poorly understood. The intrinsically disordered TSSC4 protein has been identified as a nuclear-localized U5 snRNP and U4/U6-U5 tri-snRNP assembly/recycling factor, but how TSSC4's intrinsic disorder supports TSSC4 functions remains unknown. Using diverse interaction assays and cryogenic electron microscopy-based structural analysis, we show that TSSC4 employs four conserved, non-contiguous regions to bind the PRPF8 Jab1/MPN domain and the SNRNP200 helicase at functionally important sites. It thereby inhibits SNRNP200 helicase activity, spatially aligns the proteins, coordinates formation of a U5 sub-module and transiently blocks premature interaction of SNRNP200 with at least three other spliceosomal factors. Guided by the structure, we designed a TSSC4 variant that lacks stable binding to the PRPF8 Jab1/MPN domain or SNRNP200 in vitro. Comparative immunoprecipitation/mass spectrometry from HEK293 nuclear extract revealed distinct interaction profiles of wild type TSSC4 and the variant deficient in PRPF8/SNRNP200 binding with snRNP proteins, other spliceosomal proteins as well as snRNP assembly/recycling factors and chaperones. Our findings elucidate molecular strategies employed by an intrinsically disordered protein to promote snRNP assembly, and suggest multiple TSSC4-dependent stages during snRNP assembly/recycling

A multi-factor trafficking site on the spliceosome remodeling enzyme BRR2 recruits C9ORF78 to regulate alternative splicing

The intrinsically unstructured C9ORF78 protein was detected in spliceosomes but its role in splicing is presently unclear. We find that C9ORF78 tightly interacts with the spliceosome remodeling factor, BRR2, in vitro. Affinity purification/mass spectrometry and RNA UV-crosslinking analyses identify additional C9ORF78 interactors in spliceosomes. Cryogenic electron microscopy structures reveal how C9ORF78 and the spliceosomal B complex protein, FBP21, wrap around the C-terminal helicase cassette of BRR2 in a mutually exclusive manner. Knock-down of C9ORF78 leads to alternative NAGNAG 3′-splice site usage and exon skipping, the latter dependent on BRR2. Inspection of spliceosome structures shows that C9ORF78 could contact several detected spliceosome interactors when bound to BRR2, including the suggested 3′-splice site regulating helicase, PRPF22. Together, our data establish C9ORF78 as a late-stage splicing regulatory protein that takes advantage of a multi-factor trafficking site on BRR2, providing one explanation for suggested roles of BRR2 during splicing catalysis and alternative splicing

Characterization of relativistic electron bunch duration and travelling wave structure phase velocity based on momentum spectra measurements on the ARES linac at DESY

The ARES linac at DESY aims to generate and characterize ultrashort electron bunches (fs to sub-fs duration) with high momentum and arrival time stability for the purpose of applications related to accelerator R&D, e.g. development of advanced and compact diagnostics and accelerating structures, test of new accelerator components, medical applications studies, machine learning, etc. During its commissioning phase, the bunch duration characterization of the electron bunches generated at ARES has been performed with an RF-phasing technique relying on momentum spectra measurements, using only common accelerator elements (RF accelerating structures and magnetic spectrometers). The sensitivity of the method allowed highlighting different response times for Mo and Cs2Te cathodes. The measured electron bunch duration in a wide range of machine parameters shows excellent agreement overall with the simulation predictions, thus demonstrating a very good understanding of the ARES operation on the bunch duration aspect. The importance of a precise in-situ experimental determination of the phase velocity of the first travelling wave accelerating structure after the electron source, for which we propose a simple new beam-based method precise down to sub-permille variation respective to the speed of light in vacuum, is emphasized for this purpose. A minimum bunch duration of 20 fs rms, resolution-limited by the space charge forces, is reported. This is, to the best of our knowledge, around 4 times shorter than what has been previously experimentally demonstrated based on RF-phasing techniques with a single RF structure. The present study constitutes a strong basis for future time characterization down to the sub-fs level at ARES, using dedicated X-band transverse deflecting structures.Comment: 17 pages, 11 figures. To be submitted to Physical Review Accelerators and Beam

Status and Objectives of the Dedicated Accelerator R&D Facility "SINBAD" at DESY

We present a status update on the dedicated R\&D facility SINBAD which is currently under construction at DESY. The facility will host multiple independent experiments on the acceleration of ultra-short electron bunches and novel, high gradient acceleration methods. The first experiment is the ARES-experiment with a normal conducting 100\,MeV S-band linac at its core. We present the objectives of this experiment ranging from the study of compression techniques to sub-fs level to its application as injector for various advanced acceleration schemes e.g. the plans to use ARES as a test-site for DLA experiments in the context of the ACHIP collaboration. The time-line including the planned extension with laser driven plasma-wakefield acceleration is presented. The second initial experiment is AXSIS which aims to accelerate fs-electron bunches to 15\,MeV in a THz driven dielectric structure and subsequently create X-rays by inverse Compton scattering.Comment: EAAC'17 conference proceeding

A Hybrid Spam Detection Method Based on Unstructured Datasets

This document is the accepted manuscript version of the following article: Shao, Y., Trovati, M., Shi, Q. et al. Soft Comput (2017) 21: 233. The final publication is available at Springer via http://dx.doi.org/10.1007/s00500-015-1959-z. © Springer-Verlag Berlin Heidelberg 2015.The identification of non-genuine or malicious messages poses a variety of challenges due to the continuous changes in the techniques utilised by cyber-criminals. In this article, we propose a hybrid detection method based on a combination of image and text spam recognition techniques. In particular, the former is based on sparse representation-based classification, which focuses on the global and local image features, and a dictionary learning technique to achieve a spam and a ham sub-dictionary. On the other hand, the textual analysis is based on semantic properties of documents to assess the level of maliciousness. More specifically, we are able to distinguish between meta-spam and real spam. Experimental results show the accuracy and potential of our approach.Peer reviewedFinal Accepted Versio

A multi factor trafficking site on the spliceosome remodeling enzyme BRR2 recruits C9ORF78 to regulate alternative splicing

The intrinsically unstructured C9ORF78 protein was detected in spliceosomes but its role in splicing is presently unclear. We find that C9ORF78 tightly interacts with the spliceosome remodeling factor, BRR2, in vitro. Affinity purification mass spectrometry and RNA UV crosslinking analyses identify additional C9ORF78 interactors in spliceosomes. Cryogenic electron microscopy structures reveal how C9ORF78 and the spliceosomal B complex protein, FBP21, wrap around the C terminal helicase cassette of BRR2 in a mutually exclusive manner. Knock down of C9ORF78 leads to alternative NAGNAG 3 splice site usage and exon skipping, the latter dependent on BRR2. Inspection of spliceosome structures shows that C9ORF78 could contact several detected spliceosome interactors when bound to BRR2, including the suggested 3 splice site regulating helicase, PRPF22. Together, our data establish C9ORF78 as a late stage splicing regulatory protein that takes advantage of a multi factor trafficking site on BRR2, providing one explanation for suggested roles of BRR2 during splicing catalysis and alternative splicin

The synaptic scaffold protein MPP2 interacts with GABA(A) receptors at the periphery of the postsynaptic density of glutamatergic synapses

Recent advances in imaging technology have highlighted that scaffold proteins and receptors are arranged in subsynaptic nanodomains. The synaptic membrane-associated guanylate kinase (MAGUK) scaffold protein membrane protein palmitoylated 2 (MPP2) is a component of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-associated protein complexes and also binds to the synaptic cell adhesion molecule SynCAM 1. Using superresolution imaging, we show that-like SynCAM 1-MPP2 is situated at the periphery of the postsynaptic density (PSD). In order to explore MPP2-associated protein complexes, we used a quantitative comparative proteomics approach and identified multiple γ-aminobutyric acid (GABA)(A) receptor subunits among novel synaptic MPP2 interactors. In line with a scaffold function for MPP2 in the assembly and/or modulation of intact GABA(A) receptors, manipulating MPP2 expression had effects on inhibitory synaptic transmission. We further show that GABA(A) receptors are found together with MPP2 in a subset of dendritic spines and thus highlight MPP2 as a scaffold that serves as an adaptor molecule, linking peripheral synaptic elements critical for inhibitory regulation to central structures at the PSD of glutamatergic synapses

Branch point strength controls species-specific CAMK2B alternative splicing and regulates LTP

Regulation and functionality of species-specific alternative splicing has remained enigmatic to the present date. Calcium/calmodulin-dependent protein kinase IIβ (CaMKIIβ) is expressed in several splice variants and plays a key role in learning and memory. Here, we identify and characterize several primate-specific CAMK2B splice isoforms, which show altered kinetic properties and changes in substrate specificity. Furthermore, we demonstrate that primate-specific CAMK2B alternative splicing is achieved through branch point weakening during evolution. We show that reducing branch point and splice site strengths during evolution globally renders constitutive exons alternative, thus providing novel mechanistic insight into cis-directed species-specific alternative splicing regulation. Using CRISPR/Cas9, we introduce a weaker, human branch point sequence into the mouse genome, resulting in strongly altered Camk2b splicing in the brains of mutant mice. We observe a strong impairment of long-term potentiation in CA3-CA1 synapses of mutant mice, thus connecting branch point-controlled CAMK2B alternative splicing with a fundamental function in learning and memory