Proteomics uncovers novel components of an interactive protein network supporting RNA export in trypanosomes

In trypanosomatids, transcription is polycistronic and all mRNAs are processed by trans-splicing, with export mediated by noncanonical mechanisms. Although mRNA export is central to gene regulation and expression, few orthologs of proteins involved in mRNA export in higher eukaryotes are detectable in trypanosome genomes, necessitating direct identification of protein components. We previously described conserved mRNA export pathway components in Trypanosoma cruzi, including orthologs of Sub2, a component of the TREX complex, and eIF4AIII (previously Hel45), a core component of the exon junction complex (EJC). Here, we searched for protein interactors of both proteins using cryomilling and mass spectrometry. Significant overlap between TcSub2 and TceIF4AIII-interacting protein cohorts suggests that both proteins associate with similar machinery. We identified several interactions with conserved core components of the EJC and multiple additional complexes, together with proteins specific to trypanosomatids. Additional immunoisolations of kinetoplastid-specific proteins both validated and extended the superinteractome, which is capable of supporting RNA processing from splicing through to nuclear export and cytoplasmic events. We also suggest that only proteomics is powerful enough to uncover the high connectivity between multiple aspects of mRNA metabolism and to uncover kinetoplastid-specific components that create a unique amalgam to support trypanosome mRNA maturation

Identification of a novel nucleocytoplasmic shuttling RNA helicase of Trypanosomes

Gene expression in trypanosomes is controlled mostly by post-transcriptional pathways. Little is known about the components of mRNA nucleocytoplasmic export routes in these parasites. Comparative genomics has shown that the mRNA transport pathway is the least conserved pathway among eukaryotes. Nonetheless, we identified a RNA helicase (Hel45) that is conserved across eukaryotes and similar to shuttling proteins involved in mRNA export. We used in silico analysis to predict the structure of Trypanosoma cruzi Hel45, including the N-terminal domain and the C-terminal domain, and our findings suggest that this RNA helicase can form complexes with mRNA. Hel45 was present in both nucleus and cytoplasm. Electron microscopy showed that Hel45 is clustered close to the cytoplasmic side of nuclear pore complexes, and is also present in the nucleus where it is associated with peripheral compact chromatin. Deletion of a predicted Nuclear Export Signal motif led to the accumulation of Hel45ΔNES in the nucleus, indicating that Hel45 shuttles between the nucleus and the cytoplasm. This transport was dependent on active transcription but did not depend on the exportin Crm1. Knockdown of Mex67 in T. brucei caused the nuclear accumulation of the T. brucei ortholog of Hel45. Indeed, Hel45 is present in mRNA ribonucleoprotein complexes that are not associated with polysomes. It is still necessary to confirm the precise function of Hel45. However, this RNA helicase is associated with mRNA metabolism and its nucleocytoplasmic shuttling is dependent on an mRNA export route involving Mex67 receptor

Cellular localization of tagged Hel45 in <i>Trypanosoma cruzi</i>.

<p>(A) Detection of exogenous Hel45 and a Hel45 NES deletion mutant (Hel45ΔNES) (both tagged with PTP at the NT) by indirect immunofluorescence microscopy with an anti-ProtA antibody. DAPI = DNA stained with DAPI. Hel45 =  localization of tagged Hel45 or Hel45ΔNES. MERGE = merged images for DAPI staining and Hel45 localization. N = nucleus. K = kinetoplast. Arrows = parasites with nuclear accumulation of tagged Hel45. Bar = 5 µm. (B and C) Western blot of total extract from wild-type epimastigotes (WT) and epimastigotes expressing recombinant Hel45 (B) or Hel45ΔNES (C) tagged with a PTP at the N-terminus (NT). Lane 1 =  detection with anti-Hel45 antibodies. Lane 2 =  detection with anti-ProtA antibodies.</p

Multiple sequence alignment and prediction of the structure of Hel45.

<p>(A) Multiple sequence alignment of the diagnostic conserved region of the DEAD-box helicase family (positions 25–365 according to Hel45). The nine putative conserved motifs (<i>Q</i>, <i>I</i> (WalkerA), <i>Ia, Ib, II</i> (WalkerB), <i>III, IV, V, VI</i>) are marked with orange boxes. Alignment columns displaying 100%, more than 90%, and more than 80% of similarity are highlighted in black, dark grey, and light grey, respectively. Sequences are identified with organism abbreviation and gene name, except Hel45. The organism abbreviations are: Sc: <i>Saccharomyces cerevisiae</i>, Hs: <i>Homo sapiens</i>, Pf: <i>Plasmodium falciparum</i>. The sequences have the following GenBank Identifiers (GIs): Hel45 (71418343), Sc_TIF2 (6322323), Sc_FAL1 (398365053), Sc_DBP5 (6324620), Hs_EIF4A1 (4503529), Hs_EIF4A2 (83700235), Hs_EIF4A3 (7661920), Hs_DDX19A (8922886), Pf_PFD1070w (124505577), Pf_H45 (124810293), Pf_DBP5 (6324620). (B) Schematic representation showing the nine conserved helicase motifs are boxed in orange. The N-terminal domain (NTD) contains the motifs Q, I and II for ATP-binding, Ia and Ib for RNA-binding, and III for ATP hydrolysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109521#pone.0109521-Cordin1" target="_blank">[44]</a>. The C-terminal domain (CTD) contains the motifs IV and V for RNA-binding, and VI for ATPase and unwinding activities <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109521#pone.0109521-Cordin1" target="_blank">[44]</a>. The predicted nuclear export signal (NES) in the LYDTLTI sequence (255–261 position) is shown in yellow. (C) Molecular modeling of Hel45. The nine motifs are highlighted in orange, the predicted NES (yellow) is close to the CT extremity (green). A zoom of this region (box) shows the side chains of amino-acids D257, T258 and D393, and the interactions that maintain the structure at its C-terminal extremity. The organization of the NES in the CT is shown in the inset (upper right corner).</p

Oligonucleotides used for PCR.

<p>Restriction endonuclease sites are underlined and attB recombination sites are shown in bold.</p><p>Oligonucleotides used for PCR.</p

Hel45 is a component of ribonucleoprotein complexes in the cytoplasm.

<p>Polysome fractionation by sucrose density gradient. The fractions (1–22) were collected after the sedimentation of cytoplasmic extract from <i>T. cruzi</i> treated with 100 µg/ml cycloheximide (A), 2 mM puromycin (B), or 500 U/ml micrococcal nuclease in the presence of 2 mM CaCl₂ (C). The 40S and 60S ribosomal subunits, the 80S ribosome monomer and polysomes are indicated. A western blot was performed with an anti-Hel45 antibody for each fraction. S7, a small ribosomal subunit protein, was used as a control. (D) mRNP isolation assay. Western-blot analysis with anti-Hel45 and anti-S7 antibodies and mRNPs obtained from the <i>T. cruzi</i> cytoplasmic fraction after elution from oligo(dT)-conjugated magnetic beads (El). As a control, cytoplasmic extract was treated with 10 µg/ml RNaseA before mRNP capture. FT = flow-through from cytoplasmic extract not bound to the oligo(dT). El = eluted fraction.</p

Localization of Hel45 after leptomycin B treatment in <i>T. cruzi</i> and localization of the ortholog of Hel45 (TbHel46) in <i>T. brucei</i> after Mex67 RNAi induction.

<p>(A) Detection of exogenous Hel45 tagged with PTP at the NT by indirect immunofluorescence with an anti-ProtA antibody. <i>T. cruzi</i> parasites were treated with 500 ng/ml leptomycin B (LMB) for 24 hours or were untreated (control). (B) Growth curve of <i>T. cruzi</i> parasites after treatment with 500 ng/ml leptomycin B (LMB). (C) Growth curve of <i>T. brucei</i> parasites after the induction of RNAi against Mex67 with 2 µg/ml tetracycline (RNAi-induced). (B) and (C) represent graphics of a biological replica which the density of cells in the culture was determined by counting in triplicate with a particle counter (Beckman Coulter). (D) Western-blot analysis of total protein extracts from parasites 48 (I_{48 h}) or 72 (I_{72 h}) hours after the induction of Mex67 RNAi. Non-induced (NI) parasites are shown as a control. The assay was carried out with anti-Mex67 antibodies. Anti-GAPDH antibodies were used as loading control. (E.1) Cellular localization of mRNA by fluorescence <i>in situ</i> hybridization (FISH) with a digoxigenin-conjugated oligo(dT) probe and localization of TbHel46 by indirect immunofluorescence Cells were fixed 48 hours after the induction of Mex67 RNAi (+TET). Images were processed by deconvolution software Leica AF6000. DAPI = DNA stained with DAPI. Hel45 =  localization of tagged Hel45. TbHel46 =  endogenous TbHel46 localized with anti-Hel45 antibodies. MERGE = merged images for DAPI staining and Hel45 localization (A) or DAPI staining, FISH and TbHel46 localization (E.1). N = nucleus. K = kinetoplast. (E.2 and E.3) Graphs show the quantification of fluorescence intensity of DAPI (blue), FISH (green), and TbHel46 (red) labelling that was detected across the dotted line in E.1. Fluorescence intensity was plotted in the y-axis for Mex67 RNAi-induced parasites (+TET, Figure E.2) and non-induced parasites (−TET, Figure E.3).</p

Localization of Hel45 after actinomycin D treatment in <i>T. cruzi</i>.

<p>Detection of exogenous Hel45 (A) tagged with PTP at the NT by indirect immunofluorescence with an anti-ProtA antibody and of mRNA (B) by fluorescence <i>in situ</i> hybridization (FISH) with a digoxigenin-conjugated oligo(dT) probe in <i>T. cruzi</i> after treatment with 50 µg/ml actinomycin D (ACTD) for 24 hours. Probe detection was carried out by indirect immunofluorescence with anti-DIG mouse monoclonal antibodies (Sigma-Aldrich, 1∶300 dilution) followed by secondary Alexa Fluor 488-conjugated antibodies (1∶600 dilution). As a control, 100 µg/ml RNase A was incubated with the parasites before probe hybridization (RNase A). DAPI = DNA stained with DAPI. Hel45 =  localization of tagged Hel45. MERGE = merged images for DAPI staining and Hel45 or mRNA localization. N = nucleus. K = kinetoplast. Arrows = parasites with nuclear accumulation of tagged Hel45. Bar = 5 µm.</p