The Trypanosome Pumilio Domain Protein PUF5

<div><p>PUF proteins are a conserved family of RNA binding proteins found in all eukaryotes examined so far. This study focussed on PUF5, one of 11 PUF family members encoded in the <i>Trypanosoma brucei</i> genome. Native PUF5 is present at less than 50000 molecules per cell in both bloodstream and procyclic form trypanosomes. C-terminally myc-tagged PUF5 was mainly found in the cytoplasm and could be cross-linked to RNA. PUF5 knockdown by RNA interference had no effect on the growth of bloodstream forms. Procyclic forms lacking PUF5 grew normally, but expression of PUF5 bearing a 21 kDa tandem affinity purification tag inhibited growth. Knockdown of <i>PUF5</i> did not have any effect on the ability of trypanosomes to differentiate from the mammalian to the insect form of the parasite.</p></div

PUF5-myc binds to RNA.

<p>A. Cells expressing myc-tagged PUF5-myc or UBP1-myc were UV-irradiated, protein was immunoprecipitated with anti-myc antibody, and the bound RNA was radioactively end-labelled. Samples were run on a denaturing SDS-PAGE and RNA detected by phosphorimaging. ‘*’ indicates the band corresponding to the ³²P labelled PUF5 bound RNAs while ‘**’ indicates the position of UBP1-bound RNAs. Wild type refers to the procyclic cell lines without any tagged protein. B. 10% of each sample was taken for western blot analysis using anti-myc antibody. ‘I’ refers to the input (cell lysate), ‘FT’ (flow through) refers to the unbound fraction and ‘E’ (eluate) refers to the bound fraction. Each sample represents approximately the same number of input trypanosomes.</p

<i>PUF5</i> knockdown does not affect differentiation of trypanosomes from the bloodstream to the procyclic form.

<p>Differentiation competent trypanosomes with <i>PUF5</i> RNAi were induced to differentiate into procyclic forms. This Figure shows a Western blot (top two panels) and a Northern blot (bottom two panels) for wild-type trypanosomes and two different RNAi clones, C1 and C2, after growth had resumed, but no difference was seen at any stage during the differentiation. The Western blot shows expression of EP procyclin and the Northern blot shows successful knockdown of <i>PUF5.</i> Ponceau-stained protein and methylene-blue stained rRNA serve as loading controls.</p

PUF5-myc is in the cytoplasm.

<p>An aliquot of cells expressing C-terminally myc tagged PUF5 (see Fig. 1) was used to check the localization of the protein in procyclic trypanosomes. Wild-type cells were used as the negative control. BF refers to bright field. DAPI was used to stain the nucleus and kinetoplast.</p

PUF5 is not essential for survival of PC trypanosomes.

<p>A. Cumulative growth curves of the bloodstream cells showing no difference in proliferation after <i>PUF5</i> RNAi or ectopic expression of C-terminally TAP-tagged PUF5. B. Western blot probed with anti-PUF5. C1 and C2 are different RNAi clones; their growth was indistinguishable. Ponceau S is the loading control. 5×10⁶cells loaded per lane. C. Northern blot for expression of <i>PUF5</i> RNA. Methylene-blue stained ribosomal RNA bands served as loading controls. D. Cumulative growth curves of procyclic cells ectopically expressing C-terminally myc tagged PUF5 or C-terminally TAP tagged PUF5. Results for procyclic <i>PUF5</i> double knockout cells (dKO) are also shown. E. Western blot to detect ectopic PUF5 expression, details as in (B). F. PCR amplification of a 443 bp fragment of the <i>PUF5</i> ORF in DNA from procyclic cells. A 708 bp fragment of <i>TbPUF2</i> ORF served as positive control. sKO: single knockout: dKO: double knock-out.</p

Endogenous PUF5 is not detected by a specific polyclonal antibody.

<p>Affinity purified anti-PUF5 antibody does not detect PUF5 in bloodstream-form (BS) and procyclic-form (PC) trypanosomes. Upper panel: From lanes 1 to 6, increasing amounts of recombinant protein were loaded. From lanes 7 to 11 and lanes 12 to 14, PC and BS cells were loaded, respectively. The very faint band that co-migrates with PUF5 in lanes 7, 8, 12 and 13 was background since it was also seen for the knockout (lane 9) and induced RNAi (lane 14). Lower panel: Ponceau S stain of the blot as a loading control.</p