Proteins associated with SF3a60 in T. brucei

Abstract

Trypanosoma brucei relies on Spliced leader trans splicing to generate functional messenger RNAs. Trans splicing joins the specialized SL exon from the SL RNA to pre-mRNAs and is mediated by the trans-spliceosome, which is made up of small nuclear ribonucleoprotein particles and non-snRNP factors. Although the trans spliceosome is essential for trypanosomatid gene expression, not all spliceosomal protein factors are known and of these, only a few are completely characterized. In this study, we have characterized the trypanosome Splicing Factor, SF3a60, the only currently annotated SF3a component. As expected, epitope-tagged SF3a60 localizes in the trypanosome nucleus. SF3a60 is essential for cell viability but its depletion seem to have no detectable effect on trans-splicing. In addition, we used SF3a60 as bait in a Yeast-2-hybrid system screen and identified its interacting protein factors. The interactions with SF3a120, SF3a66 and SAP130 were confirmed by tandem affinity purification and mass spectrometry.

Figure 1. Trypanosome SF3a60 localizes in the nucleus.

(A) Cells expressing SF3a60-TAP and the TAP tag with (+) and without (−) tetracycline induction after Western blot analyses. The SF3a60-TAP is conditionally expressed while tubulin (TUB) expression is used as control and (B). Immunofluorescence of procyclic form trypanosomes TAP-tagged TbSF3a60 counterstained for TbSF3a60 (red) and for DNA (DAPI).

Figure 2. S3a60 is essential for trypanosome viability.

(A) Cumulative growth curves of TbSF3a60 depleted trypanosomes. The cells grown with (triangles, solid line) or without (squares, dashed line) tetracycline. Every 24 h, samples were taken for the determination of cell density (cells/ml), and grown cultures were diluted down to 5×10⁵ cells/ml with fresh medium. (B). Effect on SF3a60 mRNA. Cells were grown either without Tet (–), or in the presence of 100 ng/ml Tet (+) for 24, 48 and 72 hours. Each lane on the Northern blot contains 10 μg RNA from bloodstream form wild type (WT) or an RNAi cell line. Asterisk (*) denotes dsRNA. Trypanosome SRP and Tubulin (TUB) RNAs are used as loading controls. Arrow indicates SF3a60 mRNA.

Figure 3. SF3a60 RNAi has no effect on splicing.

The Y structure trans splicing intermediate is not decreased after two days of RNAi targeting SF3a60. The full-length spliced leader RNA and the Y structure were detected by primer extension (diagram in panel A) followed by denaturing polyacrylamide gel electrophoresis (B) with the U3 snRNA as a loading control. The RNA was prepared from the RNAi cell line grown without tetracycline (panel B, lanes 1 and 4), or with tetracycline for one or two days (lanes 2 and 3). M: markers.

Figure 4. Preparative tandem affinity purification identifies SF3a60 associated proteins.

Proteins were separated on 12% SDS-PAGE and stained with SYPRO Ruby. Marker protein sizes in kilo Daltons are indicated on the left and corresponding protein bands are on the right. Details are in Table 1.

Figure 5. C2H2 and SWAP/Surp domains are also conserved in T. brucei SF3a subunits.

Black and light grey shading indicate identical and conserved amino acid residues, respectively. A. T. brucei, Homo sapiens (AAH11523.1) and Saccharomyces cerevisiae (CAA98589.1). Human, yeast and T. brucei have each, a C-terminal C2H2-like Zn finger motif while yeast has a second N-terminal C2H2-like motif. B. T. brucei, Homo sapiens (AAH09903.1) and Saccharomyces cerevisiae (CAA98602.1). Both human, yeast and trypanosome have a common Cys2His2-like Zn finger motif. C. T. brucei, Homo sapiens (AAH01976.1) and Saccharomyces cerevisiae (CAA89497.1). The human and yeast sequences have two SWAP/Surp domains each in tandem while T. brucei has just one SWAP/Surp domain.

Figure 6. Sequence conservation for putative T. brucei PRP40.

A. The WW domains of human FBP11 (O75400.2), yeast PRP40 (NP_012913.3) and the putative Trypanosome FBP11 homologue contain the tryptophans, prolines and a central set of three hydrophobic residues characteristic of WW domains. Evidently, the putative T. brucei FBP11 lacks a second WW domain.