RNA pull-down and mass spectrometry
Hel66 (Tb927.10.1720) was identified from a pulldown assay using the 3´ UTR of VSG121 of T. brucei. Briefly, biotinylated RNA containing the first 188 nucleotides of the VSG 3´UTR (including both the 16mer and 8mer motifs) was coupled to streptavidin beads and exposed to T. brucei cell lysate. Bound proteins were identified by mass spectrometry. Three different control RNAs were used. Control 1 was the first 188 nucleotides of the VSG 3´UTR with scrambled 16mer and 8mer, control 2 was the reverse complement of control 1 and control 3 was the first 188 nucleotides of the VSG 3´UTR with the 16mer reversed.
Cloning, expression and purification of recombinant Hel66
The DNA encoding the Hel66 open reading frame (ORF) of the T. brucei brucei strain Lister 427 was cloned into the pGEX-4T2 expression vector (GE Healthcare Life Sciences) and the plasmid transfected into ArcticExpress (DE3) E. coli cells (Agilent Technologies) for expression of Hel66 fused to an N-terminal GST-tag.
For production of recombinant Hel66 protein, 500 ml Luria-Bertani (LB) medium was inoculated with pre-cultured ArcticExpress (DE3) Hel66 cells. The cells were grown to an OD600 of ~ 0.6, cooled on ice for 30 min, induced with 0.1 mM IPTG and grown overnight at 10 ˚C. The bacteria were harvested by centrifugation (7,700 x g, 5 min, 4 ˚C), washed once with 100 ml PBS (7,700 x g, 5 min, 4 ˚C) following which the pellet was re-suspended in 50 ml of buffer A (PBS with 10 mM DTT and 1 x protease Inhibitor Cocktail (Roche)). The cells were lysed by sonication with ultrasound (10 sec pulses separated by 1 min breaks) until the solution became transparent. 1% Triton X-100 was added and the lysate was incubated for 30 min at 4˚C while rotating. The lysate was centrifuged (15,000 x g, 10 min, 4 ˚C) and the supernatant (clear lysate) was then loaded onto GST GraviTrap columns (GE Healthcare Life Sciences) that had been pre-washed with PBS and buffer B (PBS pH 7.4, 10 mM ATP, 20 mM MgCl2, 50 mM KCl, 10 mM DTT, 1 x Protease Inhibitor Cocktail (Roche)). The column was incubated for 30 min with 10 ml of buffer C (PBS pH 7.4, 10 mM DTT, 10 mM ATP, 20 mM MgCl2, 40 mM KCl, protease Inhibitor Cocktail) mixed with 23 µl of urea-denatured bacterial lysate, and then washed twice with 10 ml buffer B and twice with 10 ml buffer D (PBS pH 7.4, 5 mM ATP, 20 mM MgCl2, 50 mM KCl, 10 mM DTT, 1 x Protease Inhibitor Cocktail (Roche)). The GST-Hel66 protein was eluted with 10 ml elution buffer (50 mM Tris-Cl pH 8.0, 20 mM glutathione) and 2 ml fractions were collected. Fractions containing GST-Hel66 were identified by SDS-PAGE, pooled, concentrated and the buffer exchanged to PBS using an Amicon ultra-15 10K centrifugal filter (Merk-Millipore). The protein was frozen in liquid nitrogen and stored at -80 ˚C.
Trypanosome cell lines and culture conditions
T. brucei 13–90 cells 51 and 2T1 cells (both modified monomorphic bloodstream form Trypanosoma brucei brucei strain 427, variant MITat1.2) 52 were used throughout this study. All transgenic cell lines generated in this study are based on the 2T1 cell line. Cells were grown in HMI-9 medium 53 supplemented with 10% fetal calf serum (FCS) (Sigma-Aldrich, St. Louis, USA) and incubated at 37˚C and 5% CO2. For maintenance of previously transfected plasmids, T. brucei 13–90 cells were cultured with 5 µg/ml hygromycin and 2.5 µg/ml G418 whereas T. brucei 2T1 cells were cultured with 0.1 µg/ml puromycin and 2.5 µg/ml phleomycin. Cell numbers were monitored using either a haemocytometer or a Z2 Coulter counter (Beckman Coulter).
Plasmid construction and generation of transgenic trypanosome cell lines
The Hel66 RNAi cell line was generated using the pGL2084 vector 54 and T. brucei 2T1 cells. The conserved nature of the DExD/H protein family prevented the usage of the full open reading frame sequence of TbDHel116 for RNAi. Instead, two short fragments from the non-conserved N- and C- terminal extension regions of Hel66 were amplified with primer pairs MBS37/MBS38 and MBS39/MBS40, respectively, and then joined together in an additional PCR step using the primer pair MBS37/MBS40 to obtain a 305 bp DNA fragment with AttB adaptor sequences. This DNA fragment was cloned into pGL2084 by a BP Recombinase reaction (Invitrogen) following the manufacturer’s instructions to generate pGL2084_Hel66. The plasmid was linearised with NotI, transfected into T. brucei 2T1 cells and positive transfectants were selected with 2.5 µg/ml hygromycin. RNAi was induced with 1 µg/ml tetracycline.
For C-terminal tagging of one endogenous Hel66 allele with HA or mNeonGreen, the PCR based tagging system using pMOTag3H or pMOTag_mNG (a modified pMOTag3G plasmid with the GFP replaced by mNeonGreen) as templates, respectively, was used 55. All primers used for plasmid construction and generation of transgenic cell lines are provided in Table S1.
All transfections for generation of transgenic cell lines were carried out with 3 × 107 trypanosome cells, electroporated with 10 µg of linearised plasmid DNA or PCR product using the Amaxa Basic Parasite Nucleofector Kit 1 and Nucleofector II device (Lonza, Switzerland, program X-001).
Whole cell protein lysate from 1 x 106 trypanosome cells was separated on 12.5% sodium dodecyl sulphate (SDS)-polyacrylamide gels and transferred onto nitrocellulose membranes (GE Healthcare Life Sciences). The membranes were blocked by incubation with 5% milk powder in PBS for 1 h at room temperature (RT) or overnight at 4 ˚C. Primary antibodies (rabbit anti-VSG221, 1:5000) and mouse anti-PFR antibody (L13D6, 1:20)) 56 were then applied in PBS/1% milk/0.1% Tween-20 solution for 1 h at RT. After four washes (5 min each) with PBS/0.2% Tween-20, IRDye 800CW-conjugated goat-anti-rabbit and IRDye 680LT-conjugated goat-anti-mouse secondary antibodies were applied in PBS/1% milk/0.1% Tween-20 solution for 1 h at RT in the dark. The membranes were washed four times (5 min each in the dark) with PBS/0.2% Tween-20 followed by a final 5 min wash with PBS. Blots were analysed using a LI-COR Odyssey system (LI-COR Biosciences).
RNA extraction and Northern blot analysis
Total RNA was extracted from 1 x 108 trypanosome cells using the Qiagen RNeasy Mini Kit (Qiagen, Netherlands) following the manufacturer's instructions. Northern blot analyses were carried out using 8 µg of total RNA. The RNA was denatured with glyoxal at 50˚C for 40 min as previously described 57 and loaded on a 1.5% agarose gel containing 10 mM sodium phosphate, pH 6.9. The RNA was transferred overnight to a Hybond N + nylon membrane (GE Healthcare) by upward capillary transfer. After transfer, the RNA was UV crosslinked (1200x100 µJ/cm2) onto the membranes and deglyoxylated by baking at 80 ˚C for 1 h. The membranes were then prehybridised at 42˚C for 1 h in hybridisation solution (5x SSC (3 M NaCl, 0.3 M tri-sodium citrate, pH 7.0), 10% 50x Denhardt’s solution (1% BSA, 1% polyvinylpyrrolidone, 1% Ficoll), 0.1% SDS, 100 µg/ml heparin, 4 mM tetrasodium pyrophosphate). Membranes were hybridised overnight at 42 ˚C with hybridisation solution containing the fluorescently labelled oligonucleotide probes (10 nm each) (listed in Table S1). After hybridisation, the membranes were washed three times for 10 min with northern wash buffer (2x SSC buffer, 0.1% SDS), dried and the blots analysed using a LI-COR Odyssey system (LI-COR Biosciences).
Quantification of VSG mRNA
VSG mRNA was quantified using RNA dot blots as previously described 58. Briefly, 3 µg of glyoxal-denatured RNA was applied to a nitrocellulose membrane (Hybond-N) using a Minifold Dotblotter (Schleicher & Schuell, Germany). The blots were hybridised over night at 42 ˚C with a VSG221 oligonucleotide probe coupled to IRDye 682 and a tubulin oligonucleotide probe coupled to IRDye 782, which was used as a loading control. Blots were analysed using the LI-COR Odyssey system (LI-COR Biosciences).
The SUnSET (Surface Sensing of Translation) assay was used to monitor global translation 59. 1 x 107 mid-log phase trypanosome cells were treated with 10 µg/ml puromycin for 30 min, washed once with trypanosome dilution buffer (TDB; 5 mM KCl, 80 mM NaCl, 1 mM MgSO4, 20 mM Na2HPO4, 2 mM NaH2PO4, 20 mM glucose, pH 7.6) and boiled in 1x protein sample buffer (2% SDS, 10% glycerol, 60 mM Tris–HCl, pH 6.8, 1% β-mercaptoethanol) (5 min, 100°C). As controls, cells were either treated with 50 µg/ml of the translational inhibitor cycloheximide for 30 min prior to treatment with puromycin, or not treated with puromycin (negative control). The protein samples (containing 1 x 106 cell equivalents) were resolved on a 12.5% SDS gel and puromycin-labelled peptides were detected with anti-puromycin (1:5000 mouse anti-puromycin, clone 12D10; Sigma). Prior to antibody detection, REVERT 700 total protein stain (LI-COR Biosciences) was carried out according to the manufacturer’s instructions and used as a loading control.
RNA electrophoretic mobility shift assay (REMSA)
REMSA experiments were carried out with the Light Shift Chemiluminescent RNA EMSA kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. GST-Hel66 was incubated in reaction buffer (1x REMSA buffer, 5% glycerol, 0.1 µg/µl tRNA) containing 10 nM biotin-labelled 16mer RNA probe (Table S1) for 30 min at room temperature. For competition assays, unlabelled 16mer RNA or Biotin-IRE control RNA (RNA encoding the iron response element, provided by the kit) were added in 200-fold excess. Samples were applied to a 5% native polyacrylamide gel in 0.5x TBE (Tris-borate-EDTA) buffer. After transfer to a Hybond N + nylon membrane (GE Healthcare), samples were UV cross-linked and the biotin signal detected with HRP–conjugated streptavidin using the Chemiluminescent Nucleic Acid Detection Module (Thermo Fisher Scientific) according to the manufacturer’s instructions.
1 x 107 cells were harvested by centrifugation at 1400 x g for 10 min. The cells were washed with PBS and fixed overnight in 4% formaldehyde and 0.05% glutaraldehyde at 4 ˚C. Fixed cells were washed twice with PBS and mounted in Vectashield mounting medium with DAPI (Vector Laboratories Inc.). Images were acquired with an automated DMI6000B wide field fluorescence microscope (Leica microsystems, Germany), equipped with a DFC365FX camera (pixel size 6.45 µm) and a 100x oil objective (NA 1.4). Fluorescent images were deconvolved using Huygens Essential software (Scientific Volume Imaging B. V., Hilversum, The Netherlands) and are presented as Z-projections (method sum of slices). In order to visualise the localisation of Hel66, the DAPI signal is shown in magenta and the protein is shown in green. Image analysis was carried out using the Fiji software 60.