The eIF3 complex of Trypanosoma brucei: composition conservation does not imply the conservation of structural assembly and subunits function

Figures

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  FIGURE 1.. Subunit determination of the TbIF3-TAP complex and the TbIF3f-TAP complex. (A) Schematic drawing of a TbIF3 subunit gene in frame fusion with a MH-TAP tag at the C terminus. The MH-TAP is composed of c-myc, 6×His tag, calmodulin binding peptide, TEV protease cleavage site, and protein A as described previously (Guo et al. 2012). (B) Immunofluorescence assay of the subcellular localization of the tagged TbIF3a and TbIF3f proteins. (C) Overview of the procedures purifying the tagged TbIF3 subunit complexes and determining the protein composition. TAP, tandem affinity purification; SR staining, SYPRO Ruby staining. (D) SDS-PAGE followed by SR staining of the purified TbIF3a-TAP (left lane) and TbIF3f-TAP (right lane) complexes. Individual band was excised and used for MS determination. The names of the proteins corresponding to each band are labeled. (E) SDS-PAGE followed by silver staining of the proteins associated with the tagged TbIF3a before (−) and after RNase A (+) treatment through TAP purification.

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  FIGURE 2.. TbIF3a and TbIF3f are necessary for the growth of PF and BF T. brucei. (A) Schematic representation of the dsRNA-expressing vectors. The number represents the location of the dsRNA sequence within the coding region of TbIF3a or TbIF3f. Growth curves of PF TbIF3a-RNAi (B), BF TbIF3a-RNAi (C), PF TbIF3f-RNAi (D), and BF TbIF3f-RNAi (E) cell lines in which RNAi knockdown TbIF3a or TbIF3f was induced (I) or noninduced (NI). (F) Northern blot analysis of the RNAi knockdown efficiency with the probe against the 5′-proximal coding region of TbIF3a or TbIF3f. NI and I-48 represent RNAi noninduced and induced for 48 h, respectively. EtBr-stained rRNA was used to show the loading. The targeted mRNAs are indicated on the right. (G) Growth of PF TbIF3a RNAi-hsaIF3a TAP cell line with RNAi induced (NI) or noninduced (I). (H) Western analysis of hsaIF3a-TAP in the cell lysate from PF TbIF3a-RNAi cells or from PF TbIF3a RNAi-hsaIF3a TAP cells in which RNAi was noninduced (NI) or induced for 48 h (I-48 h) with rPAP reagent. Cumulative cell number represents the cell density multiplied by total dilution.

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  FIGURE 3.. Depletion of either TbIF3a or TbIF3f results in translation inhibition. 35S-Met was added to the PF TbIF3a-RNAi or TbIF3f-RNAi cells in which RNAi was not induced or induced for 24 or 48 h. After incubation for 60 min, the cells with equal numbers were collected and lysed. (A) The clear cell lysates were separated by SDS-PAGE followed by Coomassie blue staining (lower panel) and autoradiograph (upper panel). (B) The radioactivity of the newly synthesized proteins was measured by scintillation counting of the TCA-precipitated proteins. The assays were repeated three times and the results were shown as mean ± SD. (C) Polysome profile analysis of PF TbIF3a-RNAi and TbIF3f-RNAi cells in which RNAi was not induced (NI) or induced for 48 h (I-48 h) through sucrose-gradient centrifugation. Ninety-six fractions were taken and measured absorbance at 254 nm. Every eight fractions were combined, and RNAs were extracted and separated on agarose gel followed by EtBr staining to visualize rRNAs. The positions of 80S ribosome and polysomes are indicated.

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  FIGURE 4.. Effects of TbIF3a or TbIF3f repression on tagged TbIF3f or TbIF3a complexes, respectively. (A) Western analysis of the whole-cell lysate of PF 3a RNAi-3f TAP or 3f RNAi-3a TAP in which RNAi was not induced or induced for 48 h with rPAP reagent (upper panel). Coomassie blue staining of the membrane showed proteins load (lower panel). (B) Sedimentation profiles of the tagged-TbIF3a or -TbIF3f in the sucrose-gradient fractions by Western blot. The fractions from top to bottom are indicated. SYPRO Ruby stained gels of the TbIF3f-tag complexes (C) or the TbIF3a-tag complexes (D) purified from PF 3a RNAi-3f TAP or PF 3f RNAi-3a TAP cells via TAP purification, respectively, in which RNAi was not induced (NI) or induced for 48 h (I-48 h). Portions of SYPRO Ruby stained 8% gel are enlarged (C, right panel) to show the depletion of TbIF3a after 48 h of tet addition.

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  FIGURE 5.. CrPV IRES could mediate translation in the presence of TbIF3a and TbIF3f. (A) Schematic representation of the dual-reporter vector with different IRES between an upstream Rluc ORF and a downstream Fluc ORF. Blue line segments show the Northern blot probe specific to Rluc or Fluc. The numbers represent the location of the probe sequence within the coding region of the reporters. (B) Luciferase activity assay of PF EMCV IRES-luc, HCV IRES-luc, and CrPV IRES-luc cells. Three clones from each cell line are marked as 1, 2, and 3 randomly. The parent cell line PF29-13 was used as control. (C) Real-time RT-PCR analysis of the total RNAs from one random clone of each cell line in panel A for Rluc and Fluc mRNA's measurement. The relative reporter mRNA levels of the cell PF EMCV IRES-luc were set as one. (D) Northern blot analysis of the cell PF luc-CrPV IRES and the control cell PF 29-13, with the probe specific to Rluc (lanes 1 and 2) or Fluc (lanes 3 and 4), then stripped and rehybridized with the probe specific to α-tubulin. EtBr staining of the rRNAs is shown. (E) Luciferase activity assay of the cell lysates from TbIF3a RNAi-CrPV IRES and TbIF3f RNAi-CrPV IRES cells in which RNAi…

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  FIGURE 6.. Genome-wide UV crosslinking assay of the RNA-binding activity of TbIF3. (A) Strategy of CLiTAP. CLiTAP represents UV crosslinking and TAP purification. (B) RNA-binding activity assay of the purified TbIF3a-TAP complex through CLiTAP. The purified complexes were resolved on a 10% SDS-PAGE gel, followed by SYPRO Ruby staining (left panel) and autoradiograph (right panel). The proteins corresponding to each band are indicated.