Three putative targets of miR3 selected from the <i>Giardia</i> genomic database for testing.

a<p>- The numbering is based on the 150 nts chosen for analysis (50 nts upstream and 100 nts downstream of the stop codon).</p>b<p>- Target sites were identified with the miRanda program. The stop codon is shown in <b>bold</b>.</p

Analysis of mutations to a fully complementary (FC) miR3 target.

<p><b>A)</b> The numberings of nucleotides in FC with the seed sequence shown in red. The ΔG derived from base-pairings with miR3 is shown below. <b>B)</b> Mutations of RL-FC were carried out stepwise from the 5′-end of FC to reduce the base-pairings with miR3. Mutant transcripts were transfected into <i>Giardia</i> trophozoites along with miR3 and assayed for RLuc activity. The mean +/− SD from three independent experiments is presented. The ΔG values estimated from base-pairings with miR3 for each mutant is shown in an orange-colored line. The control column on the left represents the level of expression of each of the mutant transcripts in the absence of introduced miR3. They are each set at 100% and presented in a single column.</p

Repression of RLuc transcript expression by the miR3 seed oligomer (8 nt-Seed).

<p><b>A)</b> An 8 nt miR3 seed oligomer (8 nt-Seed) is capable of repressing expression of RL-FC by 24%, RL-FC-18 by 24.7% and RL-H2A-TS by 22.8%. <b>B)</b> The 8 nt-Seed was also tested on RL-FC and RL-FC-18 expression in the GlAgo KD cells, and showed no apparent effect. The data represent the mean +/− SD from at least two independent assays. The controls show the levels of expression of various chimeric transcripts in the absence of exogenously introduced 8nt-Seed. The values are set at 100% and presented in a single control column.</p

A Minimal Anaphase Promoting Complex/Cyclosome (APC/C) in <em>Trypanosoma brucei</em>

<div><p>The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of <i>Trypanosoma brucei.</i> In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C.</p> </div

Polyubiquitination and proteasome degradation are involved in the turnover of CycB2/cyc6 during mitosis.

<p>Cells arrested in late S-phase by hydroxyurea treatment were released for synchronous growth for 8 hours in the absence (<b>A</b>) or the presence (<b>B</b>) of 20 µM MG132. Hourly cell samples were lysed, subjected to immunoprecipitation with anti-HA mAb and fractionated with SDS-PAGE. Western blotting was used to analyze the time course of the level of CycB2/cyc6-3HA with anti-HA mAb (upper panels) or polyubiquitination staining with anti-Ubiquitin (Sigma, U5379) antibody (middle panels). Anti-Bip was used as loading controls. A quantitative analysis of CycB2/cyc6-3HA relative abundance and ubiquitin staining intensity for each experiment were plotted versus time in the bottom panels. Error bars represent the SD from two independent experiments.</p

TAP of APC1-PTP and identification of APC1-associated proteins.

<p>(<b>A</b>) Lysates of strain 427 cells transfected either with empty vector (control, lane 1) or pC.APC1.PTP (lane 2) were fractionated on 10% SDS-PAGE gel, Western blotted and simultaneously probed with anti-protein C antibody (HPC4) and anti-tubulin. The blot was then detected with an anti-mouse HRP-conjugated secondary antibody. (<b>B</b>) Stepwise Western blot monitoring of APC1-PTP during TAP. Samples were analyzed from 1. IgG input (1x), 2. IgG-Sepharose flow-through (1x), 3. The elute from IgG-Sepharose after TEV-protease digestion (5x), 4. The flow-through from anti-protein C matrix (5x), and 5. The EGTA final elute (20x). The blot was probed with HPC4 antibody and the values (in x) represent the relative amounts of samples analyzed. (<b>C</b>) Samples collected from TAP as in (<b>B</b>) were fractioned on 10% SDS-PAGE gel and stained with SYPRO-Ruby. (**) on the top indicates the position of APC1-PTP fusion protein.</p

The <i>T. brucei</i> APC/C profiles during different phases of the cell cycle progression.

<p>The final EGTA elutes from TAP of APC1-PTP from (<b>A</b>) the G1 phase enriched cells; (<b>B</b>) The late S-phase cells; (<b>C</b>) The metaphase enriched cells and (<b>D</b>) The anaphase enriched cells were each fractioned in 10% SDS-PAGE and stained with SYPRO-Ruby. The individual protein bands identified by subsequent LC-MS/MS analysis are indicated on the right-hand side of each gel panel with the molecular masses (in kDa) indicated on the left-hand side. Tub and TEV denote contaminating tubulins and TEV protease, respectively. The cell samples were also analyzed by flow cytometry and the histograms of cellular DNA contents are displayed below the corresponding SDS-PAGE panels.</p

LC-MS/MS analysis of the APC/C samples purified from different phases of <i>T. brucei</i> cell cycle progression.

<p>The number of unique peptides and percent sequence coverage are summarized across the replicate analysis as indicated. For a complete analytical report, please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059258#pone.0059258.s006" target="_blank">Table S1</a>.</p

Functional divergence between <i>T. brucei</i> and yeast APC/C subunits.

<p>Yeast complementation assay. Temperature-sensitive (<i>ts</i>) yeast mutants of APC/C subunit genes <i>apc1-1</i>, <i>cdc16-1</i>, <i>cdc23-1</i>, <i>cdc27-1</i> and <i>apc10-1</i> were transformed with pRS416-ADH plasmids expressing corresponding yeast (y) or <i>T. brucei</i> (t) full-length APC/C genes. After transformation and selection, the cloned cells were grown either at the permissive (25°C) or the restrictive (37°C) temperature. Wild-type W303 (WT) cells and yeast <i>ts</i> mutant cells transfected with the empty vector (plasmid) were used as positive and negative controls, respectively.</p

Effect of AP2 knockdown on the expression of CycB2/cyc6 in <i>T. brucei.</i>

<p>(<b>A</b>) Western blot analysis of the cell lysates of; 1. wild-type cells; and 2. the cells expressing CycB2/cyc6-3HA. The blot is stained with anti-HA mAb and the anti-tubulin antibody was used as a loading control. (<b>B</b>) The Control Cells; Cells expressing CycB2/cyc6-3HA were synchronized in late S-phase with hydroxyurea and released for synchronous growth for 8 hrs to complete a cell cycle. Hourly cell samples were lysed and fractionated in SDS-PAGE, immuno-blotted and probed with anti-HA mAB for CycB2/cyc6-3HA expression. The time course of level changes of CycB2/cyc6-3HA was quantified against tubulin control using the ImageJ software and presented in the lower panel. The relative abundance of CycB2/cyc6 has a value set at 1 from the zero time point. Error bars represent the SD from two independent experiments. (<b>C</b>) AP2 RNAi cells expressing CycB2/cyc6-3HA were induced (+Tet) for RNAi for 48 hours, and then synchronized to late S-phase with hydroxyurea and released for synchronous growth for 8 hours while the RNAi was maintained. CycB2/cyc6-3HA expression was monitored as described in (B). RT-PCR analysis of AP2 transcript levels in the hourly cell samples was performed. They and the levels of CycB2/cyc6 from the Western analysis are plotted versus time and presented in the lower left panel. Error bars represent the SD from two independent experiments. The same time samples from AP2 RNAi induced culture (+ Tet) after hydroxyurea release were analyzed by flow cytometry and the data are presented in the lower right panel.</p