Functional genomic characterization of mRNAs associated with TcPUF6, a pumilio-like protein from Trypanosoma cruzi

Abstract

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease or American trypanosomiasis. Kinetoplastid parasites could be considered as model organisms for studying factors involved in posttranscriptional regulation because they control gene expression almost exclusively at this level. The PUF (Pumilio/FBF1) protein family regulates mRNA stability and translation in eukaryotes, and several members have been identified in trypanosomatids. We used a ribonomic approach to identify the putative target mRNAs associated with TcPUF6, a member of the T. cruzi PUF family. TcPUF6 is expressed in discrete sites in the cytoplasm at various stages of the parasite life cycle and is not associated with the translation machinery. The overexpression of a tandem affinity purification-tagged TcPUF6 protein allowed the identification of associated mRNAs by affinity purification assays and microarray hybridization yielding nine putative target mRNAs. Whole expression analysis of transfected parasites showed that the mRNAs associated with TcPUF6 were down-regulated in populations overexpressing TcPUF6. The association of TcPUF6 with the TcDhh1 helicase in vivo and the cellular co-localization of these proteins in epimastigote forms suggest that TcPUF6 promotes degradation of its associated mRNAs through interaction with RNA degradation complexes. Analysis of the mRNA levels of the putative TcPUF6-regulated genes during the parasite life cycle showed that their transcripts were up-regulated in metacyclic trypomastigotes. In these infective forms no co-localization between TcPUF6 and TcDhh1 was observed. Our results suggest that TcPUF6 regulates the half-lives of its associated transcripts via differential association with mRNA degradation complexes throughout its life cycle.

FIGURE 1.

TcPUF6 is present in the ribosome-free fractions of sucrose gradients. a, the positions of the 40 and 60 S subunits, the 80 S ribosome monomer, and polysomes are indicated in the sucrose density gradient. b–d, Western blots of the collected fractions (30 μl) probed with anti-TcPUF6 (1/500) (b), anti-TcTif34 (1/500) (c), and anti-PEPCK (1/250) sera (d). Transfected parasites overexpress a TcPUF6-tagged protein. e, Western blot analysis of protein extracts from epimastigotes (lane 1), epimastigotes transfected with the pTEX-TAPTAG-transfected clones (lane 2), and the PUF6 A- and (4) B-transfected clones (lane 3), with antiserum against TcPUF6 protein (1/500 dilution). f, protein extracts were also tested with an anti-CBP serum (1/1000) (Upstate). g, the same blot was re-probed with an anti-PEPCK serum (1/500). h, immunolocalization of T. cruzi TcPUF6 in TcPUF6-TAPTAG-transfected epimastigote forms with anti-CBP antiserum (1/100). Bars, 5 μm.

FIGURE 2.

Tag affinity-purified transcripts are also present in anti-TcPUF6 immunoprecipitation elutes. Reverse transcription-PCR analysis of eluted fractions from three independent Immunoprecipitation assays using anti-TcPUF6 sera (a) or preimmune serum as a control (b). PCR products were resolved in 1.2% agarose gels. c, TcPUF6 associated mRNAs are preferentially expressed in metacyclic trypomastigotes. Total RNA from epimastigotes (white bars) and metacyclic trypomastigotes (black bars) was analyzed. Metacyclic trypomastigotes gene expression results are expressed in terms of fold change in relation to epimastigotes gene expression. The results were normalized against those for the L9 ribosomal protein. Standard deviations for triplicate experiments are shown. The numbers correspond to: Tc00.1047053508039.70 (bar 1), cytochrome b5-like (bar 2), Ser/Thr phosphatase 2A (bar 3), Tc00.1047053511523.20 (bar 4), XRND1 (bar 5), Tc00.1047053509179.50 (bar 6), PAT6 (bar 7), and 69-kDa PFR2 (bar 8).

FIGURE 3.

TcPUF6 is part of a complex bearing the TcDhh1 helicase. a, Western blot using anti-TcDhh1 antibody (1/250) of total protein input (lane 1) and eluates immunoprecipitated with preimmune (lane 2) and anti-TcPUF6 antisera (lane 3). b, Western blots with anti-TcDhh1 antibody of TAG affinity-purified proteins from vector-transfected parasites (lane 1) and TcPUF6-tagged expressing parasites (lane 2). c, Western blot using anti-TcPuf6 antibody (1/500) of total protein input (lane 1), and eluates immunoprecipitated using preimmune (lane 2) and anti-TcDhh1 antibody (lane 3).

FIGURE 4.

TcPUF6 and TcDhh1 show different co-localization patterns during the parasite life cycle. Localization of TcPUF6 (A and E) and TcDhh1 (B and F) in T. cruzi epimastigotes (A–D) and metacyclic trypomastigotes (E–H) is shown. C and G show superimposed TcPUF6 and TcDhh1 images. D and H show 4′,6′-diamino-2-phenylindole images merged with differential interference contrast images. Scale bar, 5 μm.