A5090325951- RNA Research and Splicing
- RNA and protein synthesis mechanisms
- RNA modifications and cancer
- Viral Infections and Immunology Research
- RNA Interference and Gene Delivery
- Genomics and Chromatin Dynamics
- Nuclear Structure and Function
- Fungal and yeast genetics research
- Protein Structure and Dynamics
- Neurogenetic and Muscular Disorders Research
- Cancer-related gene regulation
- RNA regulation and disease
- Plant Disease Resistance and Genetics
- Plant Molecular Biology Research
- Mass Spectrometry Techniques and Applications
- Lipid metabolism and biosynthesis
- Legume Nitrogen Fixing Symbiosis
- Biochemical and Molecular Research
- Research in Cotton Cultivation
- Inorganic Fluorides and Related Compounds
- Cellular Mechanics and Interactions
- Cancer Genomics and Diagnostics
- Cancer-related molecular mechanisms research
- Advanced Proteomics Techniques and Applications
- Polyamine Metabolism and Applications
Max Planck Institute for Multidisciplinary Sciences
2023-2024
Max Planck Institute for Biophysical Chemistry
2013-2023
Max Planck Society
2003-2013
University of Münster
2004
University of Helsinki
2004
European Molecular Biology Laboratory
2002
Philipps University of Marburg
1994-1999
Radboud University Nijmegen
1994
Columbia University
1993
Roswell Park Comprehensive Cancer Center
1989
Splicing of mammalian precursor transfer RNA (tRNA) molecules involves two enzymatic steps. First, intron removal by the tRNA splicing endonuclease generates separate 5' and 3' exons. In animals, second step predominantly entails direct exon ligation an elusive ligase. Using activity-guided purification ligase from HeLa cell extracts, we identified HSPC117, a member UPF0027 (RtcB) family, as essential subunit complex. interference-mediated depletion HSPC117 inhibited maturation...
Major structural changes occur in the spliceosome during its activation just before catalyzing splicing of pre-messenger RNAs (pre-mRNAs). Whereas small nuclear RNA (snRNA) conformation are well documented, little is known about remodeling ribonucleoprotein (snRNP) structures activation. Here, human 45S activated spliceosomes and a previously unknown 35S U5 snRNP were isolated by immunoaffinity selection characterized mass spectrometry. Comparison their protein components with those other...
Detailed knowledge of the composition and structure spliceosome its assembly intermediates is a prerequisite for understanding complex process pre-mRNA splicing. To this end, we have developed tobramycin affinity-selection method that generally applicable purification native RNP complexes. By using method, isolated human prespliceosomes are ideally suited both biochemical structural studies. MS identified >70 prespliceosome-associated proteins, including nearly all known U1 U2 snRNP...
The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization this was performed in presence heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory proteome, we isolated under low-stringency conditions using two independent methods. MS2 affinity-selected complexes supported splicing when incubated nuclear extract depleted snRNPs. Mass spectrometry identified...
The splicing factor SF3b is a multiprotein complex essential for the accurate excision of introns from pre-messenger RNA. As an integral component U2 small nuclear ribonucleoprotein (snRNP) and U11/U12 di-snRNP, involved in recognition RNA's branch site within major minor spliceosomes. We have determined three-dimensional structure human by single-particle electron cryomicroscopy at resolution less than 10 angstroms, allowing identification protein domains with known structural folds. best...
More than 200 proteins associate with human spliceosomes, but little is known about their relative abundances in a given spliceosomal complex. Here we describe novel two-dimensional (2D) electrophoresis method that allows separation of high-molecular-mass without in-gel precipitation and thus loss protein. Using this system coupled mass spectrometry, identified 171 altogether on 2D maps stage-specific complexes. By staining fluorescent dye wide linear intensity range, could quantitate...
A set of seven Sm proteins assemble on the Sm-binding site spliceosomal U snRNAs to form ring-shaped core. The U7 snRNP involved in histone RNA 3′ processing contains a structurally similar but biochemically unique core which two these proteins, D1 and D2, are replaced by Lsm10 another as yet unknown component. Here we characterize this factor, termed Lsm11, novel Sm-like protein with apparently distinct functions. In vitro studies suggest that its long N-terminal part mediates an important...
Comprehensive proteomics analyses of spliceosomal complexes are currently limited to those in humans, and thus, it is unclear what extent the spliceosome's highly complex composition compositional dynamics conserved among metazoans. Here we affinity purified Drosophila melanogaster B C formed Kc cell nuclear extract. Mass spectrometry revealed that their similar human complexes. Nonetheless, a number Drosophila-specific proteins were identified, suggesting there may be novel factors...
The deposition of proteins onto newly spliced mRNAs has far reaching consequences for their subsequent metabolism. We affinity-purified human mRNPs under physiological conditions from HeLa nuclear extract and present the first comprehensive inventory protein composition as determined by mass spectrometry. Several previously not known to be mRNP-associated were detected, including DEAD-box helicases DDX3, DDX5, DDX9, ELG, hNHN1, BCLAF1, TRAP150 proteins. association some identified mRNP was...
To better understand the compositional and structural dynamics of human spliceosome during its activation, we set out to isolate spliceosomal complexes formed after precatalytic B but prior catalytically active C complexes. By shortening polypyrimidine tract PM5 pre-mRNA, which lacks a 3′ splice site exon, stalled assembly at activation stage. We subsequently affinity purified act under same conditions previously used complexes, analyzed their protein composition by mass spectrometry. A...