Download Trypanosoma brucei - Justus-Liebig

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