1
LEGENDS TO SUPPLEMENTARY FIGURES Supplementary Figure 1. Minimum
Evolution phylogenetic tree of the SMN protein family The branches
of the tree are indicated by the NCBI GI number followed by the
abbreviated genus and species name (e.g. Homsap for Homo sapiens).
All branches are labeled with the percent value of the bootstrap
support. Two distinct phylogenetic lineages of SMN proteins are
indicated on the right side of the figure. Supplementary Figure 2.
Gemin2: multiple alignment of protein sequences ClustalW alignment
of the newly identified T.brucei Gemin2 protein with other
trypanosomatid and the human homologs. The NCBI gene identification
numbers: T.brucei (71747506, GeneDB: Tb10.70.1350), T.cruzi
(71411398), L.infantum (146103064), L.major (157876717),
L.braziliensis (154345650), H.sapiens (57165350). Secondary
structure (SS) and structural disorder predicted for T.brucei and
H.sapiens Gemin2 proteins are indicated above and below the
alignment, respectively. α-helices are represented as tubes, and
β-strands as arrows, while disordered regions are shown as ‘~’. The
number of omitted residues is indicated in parentheses. Grey shaded
dotted bar indicates the conserved Gemin2 domain. Supplementary
Figure 3. Trypanosome SMN specifically interacts with Gemin2 (A)
Specific interaction in vitro. GST-T.brucei Gemin2 (lane 3), or GST
alone as a control (lane 2), were immobilized on
glutathione-Sepharose, followed by incubation with His-tagged SMN.
After washing, bound protein was recovered and analyzed by SDS-PAGE
and Western blotting, using anti-His antibodies. For comparison,
10% of the input is shown (lane 1). The positions of protein size
markers (20 and 25 kDa) and of His-SMN are marked. (B) Specific
interaction in vivo. Extracts were prepared from a T.brucei cell
line that stably expresses PTP-tagged Gemin2 (lanes 2 and 5), as a
control from a LSm4-PTP expressing cell line. Tagged complexes were
affinity-purified by IgG Sepharose, and associated SMN protein was
detected by Western blotting with anti-SMN antibodies (lanes 1 and
2). Additional controls were His-tagged recombinant T.brucei SMN
protein detected by Western blot (lane 3), and the detection of
PTP-tagged proteins in their respective cell lines by Western
blotting with PTP-tag-specific antibodies (lanes 4 and 5). The
positions of marker proteins (in kDa) is indicated on the right.
The asterisk points to a non-specific band, the arrow to the
endogenous SMN associated with the PTP-tagged Gemin2. Supplementary
Figure 4. In vitro assembly of canonical Sm core on total RNA from
T.brucei requires all seven Sm proteins
His-tagged subcomplexes of the canonical Sm core in the following
combinations were incubated with total T.brucei RNA: two Sm
subcomplexes (as indicated, lanes 3-5) or all three subcomplexes
(lane 6). An additional control reaction included only His-SMN
(lane 2). RNA assembled in vitro into Sm cores was recovered by
His-tag pull-down and analyzed by denaturing PAGE and Northern
blotting with snRNA-specific probes, detecting U2, SL, U4, U6, U1,
and U5 snRNAs (marked on the right). 20% of the input total RNA
were analyzed for comparison (lane
2
1). The asterisks mark a degradation product of U2 or SL RNA. M,
markers (in nucleotides). Supplementary Figure 5. In vitro assembly
of canonical and U2-specific Sm cores on T.brucei U1, SL, and U2
snRNAs under competitive conditions (A-C) All four tagged Sm
subcomplexes (canonical FLAG-SmEFG, His-D1D2; His- HA-SmD3B, and
the U2-specific His-Sm16.5K/15K; for protein analysis, see lanes 2-
5) were incubated together with U1, SL, or U2 snRNA, each without
or with His-SMN (lane 1). For each reconstitution (combination of
components indicated above the lanes) a 10%-aliquot of the total
reaction (lanes 6-11) and the FLAG-pulldown material (lanes 12-17)
were analyzed for protein and RNA by SDS-PAGE and Coomassie
staining (panel A) as well as by sequential Coomassie and silver
staining (panel B; note that proteins and RNAs stain
differentially; arrows point to U1, SL, and U2 snRNAs). The
mobilities of His-SMN and the Sm proteins are indicated on the
right. In addition, 10% aliquots of each of the samples were
analyzed for the U2- specific His-Sm16.5K protein by Western
blotting, using anti-Sm16.5K antibodies (panel C). M, marker
proteins (in kDa). Supplementary Figure 6. Assembly of canonical
and U2-specific Sm heteroheptamer complexes (A) Reconstitution of
snRNA-free canonical Sm cores from the following subcomplexes:
His-tagged SmEFG (alternatively FLAG-tagged SmEFG), His-tagged
SmD1D2, and His-FLAG-tagged SmD3B (see lanes 1-4). Reconstitutions
were carried out with the combinations of subcomplexes as indicated
above the lanes, followed by FLAG-pulldown and peptide elution
(lanes 5-9), in one reaction also with RNase treatment prior to
assembly (lane 9). For each reconstitution reaction a 20%- aliquot
of the total reaction (top right panel), the FLAG-pulldown material
(middle), and 50% of the supernatant (bottom) were analyzed for
protein by SDS-PAGE. M, protein marker (in kDa). The model on the
left represents the subcomplex interactions (weak, arrow with
broken lines; strong, thick arrow) for the canonical Sm core, as
well as the replacement of the SmD3B subcomplex by Sm16.5K/15K in
the U2-specific Sm core. (B-D) Reconstitution of U2-specific Sm
cores from the following subcomplexes: FLAG-tagged SmEFG,
His-tagged Sm16.5K/15K, and His-tagged SmD1D2 (see lanes 1-3).
Reconstitutions were carried out with all three Sm subcomplexes
(lanes 4- 9), in one reaction also with RNase treatment prior to
assembly (lanes 5 and 8), and in one reaction in the presence of U2
snRNA (lanes 6 and 9). For each reconstitution reaction a
10%-aliquot of the total reaction (lanes 4-6) and the FLAG-pulldown
material (90%; lanes 7-9) were analyzed for protein by SDS-PAGE and
Coomassie staining (panel C), as well as by sequential Coomassie
and silver staining (panel B; to visualize U2 snRNA). Note that
different section of the same gel are shown in panels B and C. The
mobilities of the Sm proteins and the U2 snRNA are indicated on the
right. In panel D, 10% aliquots of each of the samples were
analyzed for the U2-specific His-Sm16.5K protein by Western
blotting, using anti-Sm16.5K antibodies. M, marker proteins (in
kDa).
Supplementary Figure 1 Palfi .et al
Supplementary Figure 2 Palfi .et al
20
25
L S
110
147
190
242
U5
U2
U4
SL
U6
U1
H is
-S M
N S
m D
1 D
0 %
M N
input (10%) FLAG pulldown (90%) 9 Sm proteins + 9 Sm proteins
+
17
17
10
17
26
His-Sm16.5K
B
C
17
26
34
43
55
M
10
15
20
His-Sm16.5KD
RNase+
SUPPLEMENTARY MATERIALS AND METHODS T.brucei cell culture, extract
preparation
Cell culture of the procyclic form of T.brucei strain 427 and of
stably transfected cell lines was done as described (Cross et al.
1991, Schimanski et al. 2004). Total cell extracts were prepared in
PA-150 buffer (150 mM KCl; 20 mM Tris– HCl, pH 7.7; 3 mM MgCl2; 0.5
mM DTT), containing a Complete Mini, EDTA-free protease inhibitor
cocktail tablet (Roche), by using a Polytron PT 3100 cell
homogenizer (Kinematica AG, Switzerland). Cell lysates were
supplemented with 0.1% Tween-20 (Sigma), and centrifuged two times
at 14,000 rpm for 15 min to remove aggregates. Tandem affinity
purification
For tandem affinity purification, the PTP tag, consisting of two
protein A domains, a TEV protease cleavage site and the protein C
epitope, was used (Schimanski et al. 2005a, 2005b). For generation
of T.brucei cell lines expressing PTP-tagged SmB, SMN or Gemin2
proteins, the open reading frames (SmB: the full ORF with 186 nts
upstream region; SMN: nucleotides 43-471 of ORF; Gemin2:
nucleotides 299-1483 of ORF) were inserted in-frame into the
pC-PTP-NEO vector upstream of the PTP tag sequence, using ApaI and
NotI restriction sites. Inserts were generated by PCR with specific
primers (for list of primer sequences, see Oligonucleotides) from
genomic DNA as template (DNAzol reagent, Invitrogen). The PTP
constructs were verified by DNA sequencing in both directions. For
genomic integration, PTP-tag plasmids were linearized inside the
open reading frames with BbsI and SalI, respectively; SmB-PTP in
the 5’-UTR region with HpaI and 10 µg of each plasmid was
electroporated, using approximately 3 X 108 T.brucei cells.
Transfected cells were selected in medium containing 40 µg/ml of
G418 (Geneticin, Gibco-BRL). Expression of PTP-tagged proteins was
analyzed by immunoblotting with PAP antibodies
(Peroxidase-Anti-Peroxidase soluble complex, Sigma), as described
under western blotting.
For a control experiment the ORF of the T.brucei U6 snRNP-specific
LSm4 protein (Tb11.01.5535) was cloned similarly into the
pC-PTP-NEO vector and expressed in T.brucei cells as PTP-tagged
LSm4 protein.
A T.brucei cell line expressing exclusively the PTP-tagged SMN
protein (SMN- PTP-EE) was generated by replacing the remaining
wild-type SMN allele of the SMN- PTP cell line with a PCR product
of the hygromycin phosphotransferase coding region fused to
SMN-specific 5' and 3' gene flanks. After electroporation, cells
were cloned by limiting dilution in the presence of G418 (40µg/ml)
and hygromycin (20µg/ml).
Tandem affinity purification of PTP-tagged proteins was done as
described (Schimanski et al. 2005a, 2005b), with minor
modifications: Briefly, T.brucei cells were collected from
2.5-liter cultures (about 4 ml packed cell volume) and lysed in 20
ml PA-150 buffer. For IgG affinity chromatography, 400-µl packed
bead volume of IgG Sepharose 6 Fast Flow beads (GE Healthcare,
Sweden) were incubated with the extracts for 2 hours at 4°C. Beads
were washed extensively in the same buffer, followed by TEV
protease buffer (PA-150 with 0.5 mM EDTA). Tagged proteins were
eluted in 1 ml of TEV protease buffer containing 100 units of AcTEV
protease (Invitrogen). For anti-ProtC affinity purification, CaCl2
was added to the eluate to a final concentration of 2 mM. The
eluate was diluted to 5 ml with PC-150 buffer (PA- 150 buffer
containing 1 mM CaCl2) and incubated for 2 hours at 4°C with 200-µl
packed bead volume of anti-protein C affinity matrix (Roche). The
beads were
2
washed with PC-150 buffer and the ProtC-tagged proteins were eluted
at RT with 0.5 ml EGTA elution buffer (5 mM Tris-HCl, pH 7.7; 10 mM
EGTA, 5 mM EDTA). Eluted proteins were precipitated with 5 volumes
of acetone, separated on 15% SDS- polyacrylamide gels (prepared
with high TEMED concentration), and stained with Coomassie
Brilliant Blue R-250. The lanes from the protein gels containing
all components of the ProtC-eluted protein samples were cut into
slices and used for mass-spectrometric (MS) analysis.
Mass-spectrometric analysis of protein samples
For MS, proteins within the gel were digested, peptides extracted
and analyzed by liquid chromatography (LC) coupled ESI-MSMS as
described (Bessonov et al. 2008). Database analysis
The accession numbers of the trypanosomatid genes are annotations
of GeneDB (http://www.genedb.org/). Protein identification of MS/MS
data was performed by Mascot
(http://www.matrixscience.com/search_form_select.html). For
similarity searches Wu-BLAST
(http://www.dove.embl-heidelberg.de/Blast2/) or NCBI BLAST
(http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) were used. Protein
sequence alignments were performed by ClustalW
(http://www.ebi.ac.uk/Tools /clustalw2/ index. html) and T-Coffee
(http://www.ebi.ac.uk/t-coffee/). Pattern and profile searches were
done by SMART (http://www.smart.embl-heidelberg.de/). Protein
structure prediction was carried out via the GeneSilico metaserver
(Kurowski and Bujnicki 2003). Phylogenetic analyses were done with
MEGA 4 (Tamura et al. 2007). Comparisons between the trypanosomal
Gemin2 candidates and families in the Pfam database (Finn et al.
2008) were done with HHsearch, a method for sequence database
searches and detection of remote homology based on the pairwise
comparison of profile hidden Markov models (HMMs; Söding et al.
2005). Recombinant proteins
Glutathione S-transferase (GST) derivatives. The ORFs of T.brucei
SMN (Tb11.01.6640), Gemin2 (Tb10.70.1350) proteins, and SMN
deletion mutants (SMN 40-157, SMN 1-121, SMN 40-121, and SMN 1-52)
were PCR-amplified from genomic DNA (for list of primer sequences
used, see Oligonucleotides) and cloned into pGEX- 2TK (full-length
proteins) or into pGEX-5X-2 vector (deletion derivatives; Amersham
Pharmacia Biotech). Constructs were expressed in Escherichia coli
BL 21 (DE3) pLysS cells, and total cell lysates were prepared by
sonication in 1X reconstitution buffer (20 mM Tris–HCl, pH 7.5, 200
mM NaCl, 5 mM MgCl2, 0.02% NP-40, 0.5 mM DTT), containing a
Complete Mini, EDTA-free protease inhibitor cocktail tablet
(Roche). Cell lysates were centrifuged at 14,000 rpm for 15 min to
remove aggregates, and GST fusion proteins were purified on
glutathione-Sepharose 4B beads (GE Healthcare, Sweden) according to
manufacturer’s protocol.
His-tag derivatives. The T.brucei SMN (Tb11.01.6640), SmB
(Tb927.2.4540) and SmD3 (Tb927.4.890) ORFs were cloned into pQE30
vector (Qiagen). Constructs were expressed in E.coli M15 [pREP4]
cells. Cell extracts were prepared by sonication in lysis buffer
(50 mM Na-phosphate, pH 8.0, 300 mM NaCl, 20 mM imidazole, 1.25
mg/ml lysozyme), followed by centrifugation at 14,000 rpm. for 15
min to remove aggregates. His-tagged proteins were purified on
Ni-NTA agarose beads (Qiagen) in 1X His-binding buffer (50 mM
Na-phosphate, pH 8.0, 500 mM NaCl, 0.02% NP-40, 20 mM imidazole),
followed by elution under native conditions (50 mM Na-phosphate, pH
8.0, 300 mM NaCl, 250 mM imidazole). Eluted proteins were
3
dialyzed against 1X Sm storage buffer (20 mM Tris-HCl, pH 7.5, 200
mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol). His-tagged T.brucei
Sm subcomplexes (SmD1D2, SmD3B, Sm16.5K/15K, SmEFG) were purified
as described (Wang et al., 2006). In each case, the first cistron
bears an N-terminal His6-tag followed by a TEV cleavage site. For
producing His-FLAG-tagged SmD3B subcomplex of T.brucei by
bicistronic expression, the ORFs of SmD3 and SmB were PCR-amplified
from the His-SmD3B-pQE30 construct (Wang et al. 2006) and cloned
into pET151/D-TOPO vector (Invitrogen). The final construct
contained a TEV cleavage site after the His- V5-tag
(His-V5-TEV-FLAG-SmD3B). For construction of the His-HA-tagged
SmD3B subcomplex the ORFs of T.brucei SmD3 and SmB were cloned as
bicistron into the pQE30 vector with an HA-tag sequence following
directly the His-tag (without TEV- cleavage site). Western blotting
The T.brucei SMN protein copurifying with PTP-tagged Gemin2 or LSm4
(as a control) was analyzed by IgG-pulldown and Western blotting
with polyclonal anti- SMN antibodies developed in rabbit (BioGenes,
Berlin). Cell lysates were prepared from T.brucei cells expressing
PTP-tagged Gemin2 or LSm4 (4ml lysate in PA-150 buffer from 1x 109
cells), then the PTP-tagged protein complexes were pulled down with
50 µl packed IgG beads, washed three times in PA-150 buffer (see
above), and once with the same buffer without 150 mM KCl. Bound
proteins were eluted by boiling in SDS gel sample buffer, separated
by 15% SDS-PAGE, and blotted to PVDF (Hybond-P, GE Healthcare).
Expression of PTP-tagged proteins was detected by incubating the
blot with PAP antibodies (Sigma, recognizing the ProteinA-part of
the tag), at a dilution of 1:2000. The presence of SMN in the
IgG-pulled down material was detected by using the antibody
recognizing the T.brucei SMN protein (described above): the blot
was reacted with the affinity-purified antibody at a dilution of
1:200, and developed by ECL (Supplementary Figure 3).
Protein–protein interaction assays by GST pulldown
For in vitro SMN-Sm protein binding, 5 µg of GST-SMN, GST-Gemin2,
or GST alone (as negative control) were immobilized on 25 µl packed
glutathione-Sepharose 4B beads and incubated with 200 pmol of
purified His-SmB, -SmD3 proteins, or His- tagged Sm-subcomplexes
(His-SmD1D2, His-FLAG-SmD3B, His-SmEFG, His- Sm16.5K/15K; only the
first protein of each subcomplex is tagged) in 500 µl of 1X
reconstitution buffer (composition is described under GST
derivatives). After a 2-hour incubation at 4°C, the beads were
washed in the same buffer (3 x 1 ml), and the bound proteins were
released by boiling in SDS-PAGE sample buffer. Eluted proteins were
resolved by 15% Tricine-SDS polyacrylamide gel electrophoresis
(Schägger and Jagow 1987), and detected by Coomassie-staining. The
interaction of His-SMN protein with GST-Gemin2 was detected by
Western blotting with penta-His mouse monoclonal antibodies
(Qiagen).
For interaction assays with SMN deletion mutants and SmD3B, ~300 ng
of GST-SMN, GST-SMN 40-157, GST-SMN 1-121, GST-SMN 40-121, GST-SMN
1-52, or GST alone (as negative control) were immobilized on 25 µl
packed glutathione- Sepharose 4B beads and incubated with 1 nmole
of purified His-tagged SmD3B subcomplex in 500 µl of 1X binding
buffer (300 mM KCl, 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 5mM DTT,
0.05% NP-40). After a 1-hour incubation at room temperature, the
beads were washed in the same buffer, and the bound proteins were
released by boiling in SDS-PAGE sample buffer. Eluted proteins were
resolved by
4
electrophoresis in a 15% SDS polyacrylamide gel, and detected by
Coomassie staining. In vitro transcription
Full-length, α-32P-UTP-labeled or un-labeled trypanosome U2 snRNA
(TbU2- WT) was transcribed in vitro by T7 RNA polymerase (MBI,
Germany), using plasmid DNA linearized by XbaI as template (Cross
et al. 1991). T.brucei full-length wild-type U1, U5 and SL RNAs
(TbU1-WT, TbU5-WT and TbSL-WT) and Sm mutant U1 and U5 snRNAs
(TbU1-mutSm and TbU5-mutSm) were transcribed by SP6 RNA polymerase
(New England Biolabs, USA) using PCR fragments as template,
generated from trypanosome genomic DNA with specific primers (see
Oligonucleotides). In both TbU1- and TbU5-mutSm RNA, the Sm site
ACUUUG was changed to ACAAAG (mutated positions underlined). A
101-nts control RNA (SLC2A2s) was also transcribed from a PCR
fragment by T7 polymerase.
α-32P-CTP-labeled TbU4-3′ half wild type RNA [TbU4-3′ half WT
(69–110)], which contains nucleotides 69–110 of the T.brucei U4
snRNA with the wild-type Sm site sequence, was transcribed by T7
RNA polymerase, using as a template two complementary DNA
oligonucleotides hybridized together. A mutant derivative, TbU4-3′
half Sm mutant RNA [TbU4-3′ half mutSm (nts 69-110)] was produced
similarly, with the Sm site AGUUUG changed to AGAAAG (mutated
positions underlined). All transcripts were uncapped.
Reconstitution of Sm cores under competitive conditions
For reconstitution of Sm cores under competitive conditions 200
pmol of each of His-SmD1D2, His-HA-SmD3B, FLAG-SmEFG and
His-Sm16.5K/15K subcomplexes (only a single subcomplex FLAG-tagged)
and FLAG-tag pulldown assays were used (Supplementary Figure 5).
All four subcomplexes were combined in 1X reconstitution buffer (as
above, without NP-40) with 100 pmol of full-length TbU1-WT, TbU2-WT
or TbSL-WT snRNAs in the presence or absence of 100 pmol His-SMN
protein in 50 µl reactions. The samples were incubated at 30 °C for
30 min, then at 37°C for 15 min. Reconstituted Sm complexes were
pulled down for 2 hours at 4°C with 25µl (packed volume) anti-FLAG
beads (M2 Agarose, Sigma), washed three times in 1 ml 1X
reconstitution buffer (containing 0.02% NP-40) and eluted by two
sequential 30-min incubations at room termperature with 3 x FLAG
peptide (Sigma; 200 ng/µl in 1X reconstitution buffer + 0.02%
NP-40). Eluted proteins and RNAs were analyzed by 15% SDS-PAGE and
sequential Coomassie and silver staining.
Control reconstitution reactions (Supplementary Figure 6) of two or
three canonical Sm subcomplexes without RNA, and of the U2-specific
Sm core without U2 snRNA, followed by FLAG pulldowns, were done as
above, using 150 pmol of each subcomplex per 1X reaction and
containing in each combination only a single FLAG- tagged Sm
protein. Potential residual amounts of RNA were removed by a 30-min
incubation at 37°C with 100 units of RNase A and T1.
For detection of the U2-specific Sm16.5K protein in the
FLAG-pulled-down material, the proteins were blotted to PVDF
membrane (like described under Western blotting) incubated with a
rabbit polyclonal anti-Sm16.5K antibody (at a dilution of 1:750;
Palfi and Bindereif 1992), and the blot was developed by ECL
(Supplementary Figures 5C and 6D).
5
Oligonucleotides DNA oligonucleotides (Sigma-Aldrich, Germany) are
listed in the following: For preparing DIG-labeled snRNA-specific
probes SP6-TbU1-Fw: 5’-ATT TAG GTG ACA CTA TAG AAC TCA CCT GCA GTG
CGT-3’; TbU1-Rev: 5’-AGG GAC GCT TTC GTT CCC-3’; TbU2-Fw: 5’-ATA
TCT TCT CGG CTA TTT AGC-3’; TbU2-Rev: 5’-ACC GTC GCG CTC CAT CC-3’;
TbU4-Fw: 5’-AAG CCT TGC GCA GGG AGG-3’; TbU4-Rev: 5’-TAC CGG ATA
TAG TAT TGC AC-3’; TbU6-Fw: 5’-GGA GCC CTT CGG GGA CA-3’; TbU6-Rev:
5’-AAA AGC TAT ATC TCT CGA AGA T-3’; SP6-TbU5-Fw: 5’-ATT TAG GTG
ACA CTA TAG GCA TCG CCG TCT CGA CTT TTA-3’; TbU5-WT-Rev: 5’-GAC ACC
CCA AAG TTT AAA CG-3’; SP6-TbSL-Fw: 5’-ATT TAG GTG ACA CTA TAG AAC
TAA CGC TAT TAT TAG AAC AG-3’; TbSL-Rev: 5’-AAA GAG TGG AGG TCA TCC
G-3’. For cloning into pC-PTP-NEO vector (ApaI and NotI sites
underlined) PTP-SmB-Fw: 5’-ATG GGC CCT CAC ACC CTA CAG CAG AA -3’,
corresponding to nucleotides -186 to -169 upstream of T.brucei SmB
gene; PTP-SmB-Rev: 5’-GAT CAG CGG CCG CGC GCG TTT CCG CTT GGC T-3’,
complementary to nucleotides 309 to 325 of T.brucei SmB gene;
PTP-SMN-Fw: 5’-ATG GGC CCT TCA CAC GAG GTG CAG GC-3’, corresponding
to nucleotides 43 to 60 of T.brucei SMN gene; PTP-SMN-Rev: 5’-GAT
CAG CGG CCG CGC TCC ACG AGC ACG CTT TC-3’, complementary to
nucleotides 452 to 471 of T.brucei SMN gene; PTP-Gem2-Fw: 5’-ATG
GGC CCA AGT ATT GCA ACA ACC GGT GA-3’, corresponding to nucleotides
299 to 319 of T.brucei Gemin2 gene; PTP-Gem2-Rev: 5’-GAT CAG CGG
CCG CGG CGG AAC CAA ACG ATT ACC ATT- 3’, complementary to
nucleotides 1461 to 1483 of T.brucei Gemin2 gene. For cloning into
pGEX-2TK vector (BamHI and EcoRI sites underlined) GST-SMN-Fw:
5’-GCA TAT GGA TCC GTC CGG CGG AAT AAT AAG TC-3’; GST-SMN-Rev:
5’-GCA TAT GAA TTC CTC TCC ACG AGC ACG CTT TC-3’; GST-Gem2-Fw:
5’-GCA TAT GGA TCC GAA GAC GAT GCT GAT GCC TAC-3’; GST-Gem2-Rev:
5’-GCA TAT GAA TTC CAG CGG AAC CAA ACG ATT AC-3’. For cloning into
pGEX-5X-2 vector (BamHI and XhoI sites underlined) GST-SMN-Fw:
5’-GCA TAG GAT CCG TCC GGC GGA ATA ATA AG-3’; GST-SMN-Rev: 5’-GCA
TAC TCG AGT TAC TCT CCA CGA GCA CGC-3’; GST-SMN-52-Rev: 5’-GCA TAC
TCG AGT CAT GGT GCT TCA TCT TCT GCC-3’; GST-SMN-40-Fw: 5’-GCA TAG
GAT CCG AGG ACC AAT GTG AAA AGG C-3’; GST-SMN-121-Rev: 5’-GCA TAC
TCG AGG TCA GCG GGA AGT CTG TC. For cloning into pQE30 vector
(restriction sites underlined) His-SMN-Fw (BamHI): 5’-ATA TGG ATC
CGT CCG GCG GAA TAA TAA GTC-3’; His-SMN-Rev (SacI): 5’-ATA TGA GCT
CTT ACT CTC CAC GAG CAC GCT-3’; His-SmB-Fw (BamHI): 5’-ATA TGG ATC
CGG CCA CCA AAA TAT GCT TCA CAA- 3’;
6
His-SmB-Rev (HindIII): 5’-ATA TAA GCT TTC AAT CGC GTT TCC GCT TGG
C-3’; His-SmD3-Fw (BamHI): 5’-ATA TGG ATC CAA CAC GGA GGG GCT CCC
G-3’; His-SmD3-Rev (HindIII): 5’-ATA TAA GCT TTT ACT TCT TTG GCT
TCT TAC GG-3’. For cloning into pET151/D-TOPO vector
TOPO-FLAG-SmD3-Fw: 5’-CAC CGA CTA CAA AGA CGA TGA CGA CAA GAA CAC
GGA GGG GCT CCC GCT-3’; TOPO-SmB-Rev: 5’-TCA ATC GCG TTT CCG CTT
GG-3’. For generating templates for transcription (Sm sites
underlined) SP6-TbU1-Fw: 5’-ATT TAG GTG ACA CTA TAG AAC TCA CCT GCA
GTG CGT-3’; TbU1-WT-Rev: 5’-AGG GAC GCT TTC GTT CCC-3’;
TbU1-mutSm-Rev: 5’-AGG GAC GCT TTC GTT CCC ACT CTT TGT TTA-3’;
SP6-TbU5-Fw: 5’-ATT TAG GTG ACA CTA TAG GCA TCG CCG TCT CGA CTT
TTA-3’; TbU5-WT-Rev: 5’-GAC ACC CCA AAG TTT AAA CG-3’;
TbU5-mutSm-Rev: 5’-GAC ACC CCT TTG TTT AAA CG-3’; SP6-TbSL-Fw: 5’-
SP6-TbSL-Fw: 5’-ATT TAG GTG ACA CTA TAG AAC TAA CGC TAT TAT TAG AAC
AG-3’; TbSL-Rev: 5’-AAA GAG TGG AGG TCA TCC G-3’. T7-TbU4-3′ half
-WT-Fw: 5’-TAA TAC GAC TCA CTA TAG GTA CTC CTT CGG GGA AAG TTT GCT
ACC CAC CAC GGG TGG GA-3’, corresponding to nucleotides 69 to 110
of wild-type T.brucei U4 snRNA; T7-TbU4-3′ half-WT-Rev: 5’-TCC CAC
CCG TGG TGG GTA GCA AAC TTT CCC CGA AGG AGT ACC TAT AGT GAG TCG TAT
TA -3’, complementary to nucleotides 69 to 110 of wild-type
T.brucei U4 snRNA; T7-TbU4-3′ half-mutSm-Fw: 5’-TAA TAC GAC TCA CTA
TAG GTA CTC CTT CGG GGA AAG AAA GCT ACC CAC CAC GGG TGG GA-3’,
corresponding to nucleotides 69 to 110 of Sm mutant T.brucei U4
snRNA; T7-TbU4-3′ half-mutSm-Rev: 5’-TCC CAC CCG TGG TGG GTA GCT
TTC TTT CCC CGA AGG AGT ACC TAT AGT GAG TCG TAT TA-3’,
complementary to nucleotides 69 to 110 of Sm mutant T.brucei U4
snRNA; SLC2A2s control: Template: 5’-CAT ATC AGG ACT ATA TTG TGG
TAA GTG CAT TAT TGC ATT TCA TTC TGA AGC AGT CCA ATG ACT ACC TAC CTT
TGT CGG AAA GTA ACT CTA AAG GCG GAT GT-3’; T7-SLC2A2s-Fw: 5'-TAA
TAC GAC TCA CTA TAG GGC ATA TCA GGA CTA TAT TGT GG-3'; SLC2A2s-Rev:
5'-ACA TCC GCC TTT AGA GTT AC-3'. For cloning the SMN stem-loop
construct SMN Fw2: 5´-AGC AGA AGC TTA CGC GTC TAC GGA AGA TGA TGA
AGT GG-3´; SMN Rv2: 5´-AGC ATT CTA GAG AGC ACG CTT TCC ACC TAC-3´.
For RT-PCR assays SMN Fw-q2: 5’-GAA GAT GAT GAA GTG GCA GAG TC-3’;
SMN Rv-q3: 5’-CTC ATA ACC CGC ATT GAA GTA AG-3’; α-Tub Fw-q3:
5’-GTG CAT TGA ACG TGG ATC TG-3’; α-Tub Rv-q3: 5’-GAG AGT TGC TCG
TGG TAG GC-3’; α-Tub Fw-q1 (unspliced): 5’-GTA AGT GGT GGT GGC GTA
AG-3’; α-Tub Rv-q1(unspliced): 5’-CAA TGT GGA TGC AGA TAG
CC-3’;
7
α-Tub Rv: 5’-CTA GTA CTC CTC CAC ATC CTC CTC AC-3’; Oligo-dT18:
5’-TTT TTT TTT TTT TTT TTT-3’; 7SL RNA Rv-q2N: 5’- CTC GGT GTG CTT
CTG CAA C-3’; 7SL RNA Fw-q3: 5’-TGA CTT GGT GTT CTG CTT GG-3’; 7SL
RNA Rv-q3: 5’-TCG GTG TGC TTC TGC AAC-3’; 7SL RNA Fw-q4: 5’-GTT GCG
TTG ACT TGG TGT TC-3’; SL 6-28: 5’-ACG CTA TTA TTA GAA CAG TTT
CT-3’; PAP Igr Fw1: 5’-CCT CCT CCA CTT TCC TAC GC-3’; PAP Iorf Rv1:
5’-GTT TCG TTG GGC CAT ACA TC-3’; PAP Iorf Fw1: 5’-CCT ACC CAT TTG
GTT CAT GC-3’; PAP Int Rv1: 5’-GAA GAG GAC GGG AGA AGA GC-3’; PAP
Iorf Rv2: 5’-GGA ACT CTG GCA GCG ACT AC-3’; ATP Hel Iorf Fw1:
5’-GCG GGC TTG ACA TTA AGA AC-3’; ATP Hel Int Rv1: 5’-CGT TGT GGA
ATG TGC CTA TG-3’; ATP Hel Int Fw1: 5’-CCG TTG CTC TCA TTG TGA
TG-3’; ATP Hel Iorf Rv2: 5’-TGG TGG AAT CTC CTG ATT GG-3’; PPIase
Iorf Rv1.S: 5’-CGT TGC GAC CAC TTC TGC A-3’; PRP8 Igr Fw1: 5’-TCC
GTG TTT CTG TTT GCC TA-3’; PRP8 Iorf Rv1: 5’-GCT CAA AGC CAT CCT
CTG TC-3’; PRP8 Iorf Fw1: 5’-CAA ACG GAG GGA CTC ACA AC-3’; PRP8
Iorf Rv2: 5’-TTC CAT CCA TTG TCT GTT GG-3’; PPIase Iorf Fw1: 5’-GTC
CGA AAA GCT GAG AGC AG-3’; Gem2 Fw-q2: 5’-GGC ATT ACC GCT CTC TTC
AC-3’; Gem2 Rv-q3: 5’-CTG TCA ACG CAC TCG TCT TC-3’. SUPPLEMENTARY
REFERENCES Bessonov, S., Anokhina, M., Will, C.L., Urlaub, H., and
Lührmann, R. 2008. Isolation
of an active step I spliceosome and composition of its RNP core.
Nature 452: 846- 850.
Cross, M., Günzl, A., Palfi, Z., and Bindereif, A. 1991. Analysis
of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei:
structural organization and protein components of the spliced
leader RNP. Mol. Cell. Biol. 11: 5516-5526.
Finn, R.D., Tate, J., Mistry, J., Coggill, P.C., Sammut, S.J.,
Hotz, H.R., Ceric, G., Forslund, K., Eddy, S.R., Sonnhammer, E.L.,
and Bateman, A. 2008. The Pfam protein families database. Nucleic
Acids Res. 36: D281-D288. Database issue.
Kurowski, M.A. and Bujnicki, J.M. 2003. GeneSilico protein
structure prediction meta- server. Nucleic Acids Res. 31:
3305-3307.
Palfi, Z. and Bindereif, A. 1992. Immunological characterization
and intracellular localization of trans-spliceosomal small nuclear
ribonucleoproteins in Trypanosoma brucei. J. Biol. Chem. 267:
20159-20163.
Schägger, H. and von Jagow, G. 1987. Tricine-sodium dodecyl
sulfate- polyacrylamide gel electrophoresis for the separation of
proteins in the range from 1 to 100 kDa. Anal. Biochem. 166:
368-379.
Schimanski, B., Laufer, G., Gontcharova, L., and Günzl, A. 2004.
The Trypanosoma brucei spliced leader RNA and rRNA gene promoters
have interchangeable TbSNAP50-binding elements. Nucleic Acids Res.
32: 700-709
Schimanski, B., Nguyen, T.N., and Günzl, A. 2005a. Characterization
of a multisubunit transcription factor complex essential for
spliced-leader RNA gene transcription in Trypanosoma brucei. Mol.
Cell. Biol. 25: 7303-7313.
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Schimanski, B., Nguyen, T.N., and Günzl, A. 2005b. Highly efficient
tandem affinity purification of trypanosome protein complexes based
on a novel epitope combination. Eukaryot. Cell 4: 1942-1950.