Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Affinity purification of RNA-protein complexes from Trypanosoma brucei – principles and examples Dr. Christian Preußer Justus-Liebig-Universität Gießen Fachbereich Biologie und Chemie Institut für Biochemie AG Bindereif Trypanosoma brucei Trypanosoma brucei Sleeping sickness Nagana disease Trypanosoma brucei (15-40µm; 1-2µm) • Genome sequenced (Berriman et al. 2005) • Classical model organism • RNA editing, Variant surface glycoprotein, Polycistronic gene organization, Spliced Leader trans splicing Trans-splicing Gene expression in trypanosomes The Spliceosome • Catalyzes the removal of introns and the ligation of the flanking exons • Ribonucleoprotein complex (>3 MDa) • Composed of: – 5 small nuclear ribonucleoprotein complexes, called snRNPs – a range of non-snRNP associated splicing factors • Each snRNP consists of: – Uridine rich snRNA with defined secondary structure – Set of common and snRNP specific proteins • T.brucei snRNPs: – SL, U1, U2, U4, U5 and U6 snRNP (names according to snRNAs) Tandem affinity purification (TAP) • TAP (Tandem affinity purification) • Native, non-denaturing purification of proteins / RNA-protein complexes • ~ 18 kDa, originally developed in the yeast system • Development: PTP tag (protein C TEV protein A), pC-PTP-NEO vector • Composed of: – 2 protein A domains – TEV cleavage site – Protein C epitope Tandem affinity purification (TAP) Tandem affinity purification (TAP) TAP tag protein C TEV 2 X protein A IgG selection (= first step) snRNP protein 3` flank TEV cleavage transfect T.brucei, genomic integration stably transfected cell line anti-protein C purification (= second step) EGTA native complexes cell extract affinity purification ● RNA analysis, ● protein analysis (mass spectrometry) ● functional studies Tandem affinity purification (TAP) - example 15% SDS-SPAGE Coomassie Theoretical experiment • Usage of the TAP tag procedure to purify snRNA-protein complexes from T.brucei – Cloning steps: Incorporate gene of interest (here SmB) in pC-PTP-NEO vector – Linearize plasmid – Electroporation, homologous recombination and selection – Extract from cell line that stably express PTP tagged SmB – After IgG purification (first step) associated snRNAs are analyzed by Northern blotting (mixed DIG probe for all snRNAs) Schematic display of the Northern blot detection The U1 snRNP of Trypanosoma brucei U1C U1-70K 21.7 kDa Protein 32 kDa Protein N-terminal C2H2 type Zinc finger motif within the first highly conserved 40 amino acids N-terminal RNA recognition motif (RRM) highly basic N-terminal extension before the ZF motif interacts with the 5’-terminal single-stranded region of U1 snRNA represents a minimal version of 70K, compared with its human ortholog recognizes the loop nucleotides of the U1 snRNA iCLIP – individual crosslink and immunoprecipitation iCLIP – statistics U1C U1-70K iCLIP – U1C crosslink profile on the U1 snRNA 5‘ splice site recognition by U1C U1C is not required for parasite viability RT-PCR qRT-PCR Western blot U1C is required for efficient cis-splicing RT-PCR