The trypanosome Pumilio-domain protein PUF7 associates with a nuclear cyclophilin and is involved in ribosomal RNA maturation

Abstract

Proteins with Pumilio RNA binding domains (Puf proteins) are ubiquitous in eukaryotes. Some Puf proteins bind to the 3'-untranslated regions of mRNAs, acting to repress translation and promote degradation; others are involved in ribosomal RNA maturation. The genome of Trypanosoma brucei encodes eleven Puf proteins whose function cannot be predicted by sequence analysis. We show here that epitope-tagged TbPUF7 is located in the nucleolus, and associated with a nuclear cyclophilin-like protein, TbNCP1. RNAi targeting PUF7 reduced trypanosome growth and inhibited two steps in ribosomal RNA processing.

Fig. 1

(A) Phylogenetic tree of possible homologues of S. cerevisiae Puf6 and Nop9. Accession numbers for all species apart from T. brucei and S. cerevisiae are shown. Each protein is designated according to the sequence sued for the BLASTp search – for example, TpPuf6 was the best match to ScPuf6. protein used for the Species abbreviations are: Tt – Tetrahymena thermophila, Pf – Plasmodium falciparum; Gi – Giardia intestinalis; Gl – Giardia lamblia; Dd – Dictyostelium discoideum; Tp – Thalassiosira pseudonana; A – Arabidopsis thaliana (official gene designations given); Hs – Homo sapiens; Tv – Trichomonas vaginalis (here there were several matches, only the best is shown). Note that the Dictyostelium Nop9 sequence gave the putative human Nop9 as the best match in the human genome, but did not give a significant match to S. cerevisiae. The tree was created in DNAStar using ClustalW; bootstrap values are from 1000 trials with a seed of 111. (B) Western blot for a procyclic trypanosome line with V5 tagged PUF7, with RNAi against PUF7. The blot was probed with antibody to the V5 tag, and to aldolase (ALD) as loading control. WT: trypanosomes expressing the tet repressor but without an RNAi construct. (C) Quantitation of PUF7 RNA and PUF7 protein after RNAi. Results are for trypanosomes without an RNAi plasmid (WT) and two experiments for the RNAi line.

Fig. 2

PUF7 is in the nucleolus. Trypanosomes expressing (A) PUF7-TAP or (B and C) V5-PUF7 were stained with antibodies and with DAPI as shown. Secondary antibodies were Alexa594 (A and B) and (C) Alexa 488 (pol I) and Alexa 568 (V5). The cell cycle stages of the parasites, judged from the numbers of nuclei and kinetoplasts, are indicated.

Fig. 3

RNAi targeting PUF7 inhibits rRNA processing. (A) Schematic overview of rRNA processing in trypanosomes . (B) Total RNA was prepared from cells with no RNAi (lane 3), RNAi targeting PUF7 (lanes 4, 5, 7 and 8) or RNAi targeting NMD3 (lanes 1 and 2). To induce RNAi the cells were grown for 48 h with tetracycline (lanes 2, 5 and 8). After denaturing agarose gel electrophoresis, RNA was blotted and stained with methylene blue. LSU: large subunit rRNA; SSU: small subunit rRNA; M: markers. (C) The blot was hybridised with a probe located 3′ of the mature SSU rRNA. (D) The intensities for the pre-rRNA, 3.4 kb, and 2.6 + 2.5 kb bands are expressed as mean ± standard deviation. Sample numbers are shown above the columns. The results shown are for procyclic cells without V5-tagged PUF7; in an experiment with the V5-tagged line, in which V5-PUF7 was decreased only two-fold, an increase in the 9.2 kb band was also seen. Using a Students t-test, the induced RNAi lines were statistically significant from the uninduced lines at the following levels: (a) <0.01; (b) <0.05; (c) <0.1.

Fig. 4

PUF7 is associated with a cyclophilin-like protein. (A) NCP1-myc was expressed in trypanosomes with or without V5-tagged PUF7. Lysates were immunoprecipitated with anti-V5 antibody. The lysates, unbound and bound fractions were separated by SDS–PAGE, blotted and probed with an anti-myc antibody to detect NCP1-myc. The relative loading is indicated above the lanes. (B) As for (A), but using trypanosomes expressing V5-PUF7 with or without NCP1-myc, and precipitated with anti-myc antibody before probing to detect V5-PUF7. (C) NCP1 is in the nucleus. NCP1-myc was inducibly expressed and detected with anti-myc antibodies, with DAPI to stain DNA.