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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
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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
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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
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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
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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
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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).
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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’;
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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’;
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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.
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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.