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

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

<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

Embryonic Stem Cells Exhibit mRNA Isoform Specific Translational Regulation

<div><p>The presence of multiple variants for many mRNAs is a major contributor to protein diversity. The processing of these variants is tightly controlled in a cell-type specific manner and has a significant impact on gene expression control. Here we investigate the differential translation rates of individual mRNA variants in embryonic stem cells (ESCs) and in ESC derived Neural Precursor Cells (NPCs) using polysome profiling coupled to RNA sequencing. We show that there are a significant number of detectable mRNA variants in ESCs and NPCs and that many of them show variant specific translation rates. This is correlated with differences in the UTRs of the variants with the 5’UTR playing a predominant role. We suggest that mRNA variants that contain alternate UTRs are under different post-transcriptional controls. This is likely due to the presence or absence of miRNA and protein binding sites that regulate translation rate. This highlights the importance of addressing translation rate when using mRNA levels as a read out of protein abundance. Additional analysis shows that many annotated non-coding mRNAs are present on the polysome fractions in ESCs and NPCs. We believe that the use of polysome fractionation coupled to RNA sequencing is a useful method for analysis of the translation state of many different RNAs in the cell.</p></div

Polysomal association of ncRNA in ESC and NPC.

<p>(A,B) Analysis of the distribution of annotated non-coding RNAs in the different polysome fractions of ESCs and NPCs. (C) Polysome profiles of ESCs, NPCs and puromycin treated ESCs (PURO) to selectively disrupt polysomes. (D) qRT-PCR of representative non-coding RNAs showing their distribution in polysome gradients from ESCs and NPCs with and without the addition of puromycin.</p

Polysome profiling of ESCs and NPCs.

<p>(A) Efficiency of NPC differentiation. Mouse <i>Sox1-GFP</i> ESCs were differentiated to NPCs for 6 days. Flow cytometry of the SOX1-GFP signal showing over 80% SOX1-GFP positive cells after differentiation. (B) ESCs and NPCs were subjected to polysome profiling and 12 fractions were collected into four groups. No translation F1-5), Low translation (F6-8), High translation (F9-11) and the bottom of the gradient (F12).</p

Transcriptional and translational changes upon differentiation of ESCs to NPCs.

<p>(A) Pie chart showing the degree of translational shift of mRNAs during ESC to NPC differentiation. 5% of mRNAs display a translation shift greater than 20% upon NPC. (B) Pie chart showing transcriptional changes in the sub-fraction of translationally regulated genes. 58% of these genes are purely regulated translationally and show less than 2 fold changes in total mRNA levels. (C) Real-time PCR analysis of <i>Tchp</i> showing increased enrichment in the heavy polysome fractions in NPCs. (D) Western blot of TCHP protein in ESCs and NPCs. GAPDH is shown as a loading control.</p