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First published online September 22, 2005
doi: 10.1242/10.1242/jcs.02578

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The Trypanosoma brucei signal recognition particle lacks the Alu-domain-binding proteins: purification and functional analysis of its binding proteins by RNAi

Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel

View larger version (37K): [in a new window]   Fig. 1. Sequence alignment of T. brucei SRP19 with its homologues. (A) The alignment was performed using the ClustalW multiple sequence alignment program (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalwan.html). Residues red, green and blue represent identity, similarity and weak similarity, respectively. Pink represents residues that are conserved between trypanosome species. Red circles indicate indirect contact sites between SRP19 and helix 6 that are conserved in M. jannaschii and H. sapiens. Blue squares represent indirect contact sites between M. jannaschii SRP19 and helix 8. Secondary structure elements are shown underneath the sequence. The sequences were obtained from the following sources: TbSRP19, T. brucei, GeneDB acc. no. Tb10.1560; TcSRP19, T. cruzi, GeneDB acc. no. Tc00.1047053511281.59; LmSRP19, L. major, GeneDB acc. no. LmjF19.1600; HsSRP19, Homo sapiens, TrEMBL acc. no. P09132; MjSRP19, M. jannaschii, TrEMBL acc. no. Q58440. (B) Schematic representation of the T. brucei SRP S domain. The sequence of the 7SL RNA S domain is from Michaeli et al. (Michaeli et al., 1992). The helices and loops are designated by H and L, respectively, and their numbers are indicated.

View larger version (14K): [in a new window]   Fig. 2. Growth curves of cells silenced for SRP protein. (A) SRP72. (B) SRP68. (C) SRP19. The arrow indicates the time at which tetracycline was added. The growth of uninduced cells (-Tet) was compared with the induced cells (+Tet). The number of uninduced cells is designated by squares and of induced cells by circles.

View larger version (28K): [in a new window]   Fig. 3. (A) Northern analysis of SRP72, SRP68 and SRP19 mRNA upon silencing. RNA was prepared from cells carrying the RNAi construct, uninduced (-Tet) and after 2-3 days of induction (+Tet). Total RNA (20 µg) was subjected to northern analysis with random-labeled probes. (1) SRP72-depleted cells. (2) SRP68-depleted cells. (3) SRP19-depleted cells. The transcripts of SRP72, SRP68, SRP19, tubulin mRNAs, rRNA and dsRNA are indicated by arrows. (B) Western analysis of SRP19 protein. Whole cell extract (106 per lane) was prepared from cells carrying the RNAi construct, uninduced (-Tet) and after 4 days of induction (+Tet). The extract was fractioned on a 12% SDS-polyacrylamide gel and subjected to western analysis with the anti-SRP19 antibodies. SRP19 and a non-specific protein band used as a control for the amount of protein loaded are indicated by arrows.

View larger version (17K): [in a new window]   Fig. 4. The effect of SRP silencing on EP translocation. Immunofluorescence assay (IFA) with anti-EP (surface staining). Cells were fixed with 4% formaldehyde for 25 minutes and incubated with anti-EP antibodies. Uninduced (-Tet) cells, induced cells on the third day after induction (+Tet) and the type of cell lines are indicated. Bar, 5 µm.

View larger version (16K): [in a new window]   Fig. 5. The effect of SRP silencing on the size of the complex. Whole cell extracts (5x109 cells) were prepared from uninduced (-Tet) cells and from cells carrying SRP72, SRP68 and SRP19 RNAi constructs after 3 days of induction (+Tet). The extracts were layered on continuous 10-30% (w/v) sucrose gradients. Gradients were centrifuged at 4°C for 22 hours at 35,000 rpm using a Beckman SW41 rotor. RNA was prepared from the sucrose gradient fractions and subjected to northern analysis with random-labeled 7SL RNA and with oligonucleotides complementary to SL RNA probes. Fraction numbers are indicated (from top to bottom). S values were determined relative to 28S rRNA, 4S RNA and catalase (11S). The transcripts of 7SL RNA and SL RNA are indicated by arrows.

View larger version (38K): [in a new window]   Fig. 6. The effect of SRP72 and SRP68 silencing on 7SL RNA localization. (A) Localization of the 7SL RNA by biochemical fractionation. Cytoplasmic and nucleolar fractions were as described in Materials and Methods. RNA from these samples was subjected to northern analysis with random-labeled 7SL and U3 RNA probes from uninduced cells (-Tet) and cells after 3 days of induction (+Tet). (1) SRP72-depleted cells. (2) SRP68-depleted cells. (B) Localization of 7SL RNA during SRP72 and SRP68 silencing by in situ hybridization. Cells uninduced (-Tet) and after 3 days of induction (+Tet) were fixed and hybridized with DIG-labeled PCR probes to U3 and 7SL RNA. (1) Cells carrying the SRP72 RNAi construct hybridized with DIG-labeled PCR probe to 7SL RNA. (2) Cells carrying the SRP72 RNAi construct hybridized with DIG-labeled PCR probe to U3 RNA. (3) Cells carrying the SRP68 RNAi construct hybridized with DIG-labeled PCR probe to 7SL RNA. The nuclei (N) and nucleolus (Nc) are indicated. Bar, 5 µm.

View larger version (34K): [in a new window]   Fig. 7. TAP purification of SRP. (A) Fractionation of RNP particles on a sucrose gradient. Whole cell extract (150 ml culture, 107 cells/ml) was prepared from induced cells carrying the SRP19TAP construct. The extract was layered on a continuous 10-30% (w/v) sucrose gradient. The gradient was fractionated as described in Fig. 5. RNA extracted from an aliquot of odd-numbered fractions was separated on a 10% polyacrylamide-7M urea gel and subjected to northern analysis with random-labeled 7SL RNA. Fraction numbers are indicated (from top to bottom). Proteins extracted from an aliquot of even numbered fractions were separated on a 12% SDS-polyacrylamide gel and subjected to western analysis with IgG antibodies. Fraction numbers are indicated (from top to bottom). S values were determined relative to 28S rRNA, 4S RNA and catalase (11S). The transcripts of 7SL RNA and the SRP19TAP protein are indicated by arrows. (B) Immunoprecipitation of the SRP by IgG. Whole cell extract was prepared from induced cells (50 ml, 107 cells/ml) carrying the SRP19TAP construct and subjected to affinity selection using IgG Sepharose. The immunoprecipitated products and 2% of total cell extract and supernatant were separated on a 12% SDS-polyacrylamide gel and subjected to western analysis with IgG antibodies. SRP19TAP is indicated by an arrow. (C) Purification of SRP proteins. Whole cell extracts (1 litre, 107 cells/ml) were prepared from induced cells carrying the SRP19TAP construct and from uninduced cells (control) and the extracts were subjected to affinity selection using IgG Sepharose. Proteins from uninduced cells are shown in lane 1, and induced cells in lane 2. Complexes selected with IgG beads were treated with TEV protease and were further purified by DEAE column (lane 3). Proteins extracted from the eluate were fractioned on a 10% SDS-polyacrylamide gel and stained with silver. SRP21 (SRP19 homolog fused to CBP), SRP19TAP (40 kDa), SRP54, SRP64 (SRP68 homolog) and SRP75 (SRP72 homolog) are indicated by arrows. (D) Purification of SRP RNAs. As for C except that RNA was prepared from the affinity selection fraction, separated on a 10% polyacrylamide-7M urea gel and stained with silver. 7SL RNA and sRNA-76 are indicated by arrows. (E) Immunofluorescence assay (IFA) of the SRP19TAP protein. Cells carrying the SRP19TAP construct were induced for 4 hours (1) and 60 hours (2) and the tagged protein was visualized. Cells were fixed with 4% formaldehyde for 25 minutes, incubated with IgG and visualized as described in Materials and Methods. Bar, 5 µm.