CDK Phosphorylation of Translation Initiation Factors Couples Protein Translation with Cell-Cycle Transition.

Protein translation in eukaryotes is cell-cycle dependent, with translation rates more robust in G1 phase of the cell cycle than in mitosis. However, whether the fundamental cell-cycle control machinery directly activates protein translation during the G1/S cell-cycle transition remains unknown. Using the early divergent eukaryote Trypanosoma brucei as a model organism, we report that the G1 cyclin-dependent kinase CRK1 phosphorylates two translation initiation factors, eIF4E4 and PABP1, to promote the G1/S cell-cycle transition and global protein translation. Phosphorylation of eIF4E4 by CRK1 enhances binding to the m7G cap structure and interaction with eIF4E4 and eIF4G3, and phosphorylation of PABP1 by CRK1 promotes association with the poly(A) sequence, self-interaction, and interaction with eIF4E4. These findings demonstrate that cyclin-dependent kinase-mediated regulation of translation initiation factors couples global protein translation with the G1/S cell-cycle transition.

The G1 Cyclin-dependent Kinase CRK1 in Trypanosoma brucei Regulates Anterograde Protein Transport by Phosphorylating the COPII Subunit Sec31.

Transport of secretory proteins from the endoplasmic reticulum to the Golgi is mediated by the coat protein II (COPII) complex comprising a Sec23-Sec24 heterodimer and a Sec13-Sec31 heterotetramer. The mechanisms underlying COPII-mediated protein trafficking have been well defined, but the extent of regulation of this secretory machinery by cellular signaling pathways remains poorly understood. Here, we report that CRK1, a G1 cyclin-dependent kinase in Trypanosoma brucei, regulates anterograde protein trafficking by phosphorylating Sec31. Depletion of CRK1 abolished anterograde transport of the secretory protein and disrupted the localization of multiple Golgi proteins, reminiscent of Sec31 depletion. CRK1 phosphorylates Sec31 at multiple serine/threonine sites, and mutation of these phosphosites to alanine recapitulates the protein trafficking defects caused by Sec31 depletion. Mutation of these CRK1 phosphosites to aspartate restored Sec31 function. Taken together, these results uncover a novel function of CRK1 in anterograde protein trafficking and elucidate the mechanistic role of CRK1 in protein trafficking through regulation of the COPII subunit Sec31.

The microRNAs in an ancient protist repress the variant-specific surface protein expression by targeting the entire coding sequence.

microRNAs (miRNA) have been detected in the deeply branched protist, Giardia lamblia, and shown to repress expression of the family of variant-specific surface proteins (VSPs), only one of which is expressed in Giardia trophozoite at a given time. Three next-generation sequencing libraries of Giardia Argonaute-associated small RNAs were constructed and analyzed. Analysis of the libraries identified a total of 99 new putative miRNAs with a size primarily in the 26 nt range similar to the size previously predicted by the Giardia Dicer crystal structure and identified by our own studies. Bioinformatic analysis identified multiple putative miRNA target sites in the mRNAs of all 73 VSPs. The effect of miRNA target sites within a defined 3'-region were tested on two vsp mRNAs. All the miRNAs showed partial repression of the corresponding vsp expression and were additive when the targeting sites were separately located. But the combined repression still falls short of 100%. Two other relatively short vsp mRNAs with 15 and 11 putative miRNA target sites identified throughout their ORFs were tested with their corresponding miRNAs. The results indicate that; (1) near 100% repression of vsp mRNA expression can be achieved through the combined action of multiple miRNAs on target sites located throughout the ORF; (2) the miRNA machinery could be instrumental in repressing the expression of vsp genes in Giardia; (3) this is the first time that all the miRNA target sites in the entire ORF of a mRNA have been tested and shown to be functional.

Experimental verification of the identity of variant-specific surface proteins in Giardia lamblia trophozoites.

The cell membrane of a Giardia lamblia trophozoite is covered with a single species of variant-specific surface protein (VSP) that is replaced by another VSP every 6 to 13 generations of cell growth, possibly for an evasion of host immunity. Experimentally, only six VSP species have been verified to localize to the cell membrane thus far. By assuming that VSP contains multiple CXXC motifs, 219 vsp genes were annotated in GiardiaDB of the WB isolate. By further assuming that VSP possesses both CXXC motifs and a CRGKA tail at the C terminus, Adam et al. (BMC Genomics 11:424, 2010) identified a total of 303 potential vsp genes in Giardia WB. The discrepancies between these two assumed VSP identities have caused some confusion. Here, we used experimental approaches to further verify what is required of the structures of a VSP to localize to the surface of cell membrane. The data led to the following conclusions. (i) The C-terminal CRGKA sequence is not essential for localizing VSPs to the cell membrane. (ii) A "motif 1" of 45 residues, consisting of two CXXCs separated by 12 to 15 amino acid residues, located close to the C terminus and a hydrophobic "motif 2" of 38 residues at the C terminus are both essential and sufficient for localizing the protein to the cell membrane. (ii) An N-terminal sequence upstream from motif 1 is not required for targeting VSPs to the cell membrane. By these criteria, we are able to identify 73 open reading frames as the putative vsp genes in Giardia. IMPORTANCE The intestinal pathogen Giardia lamblia expresses only one variant-specific surface protein (VSP) on the cell membrane surface at a given time, but it changes spontaneously every 6 to 13 generations of growth, presumably for evading the host immunity. Only 6 VSPs have been empirically shown to localize to the cell membrane surface thus far. Here, we used mutations of VSPs and methods of identifying their locations in Giardia cells and found that a "motif 1" of 45 residues, consisting of two CXXCs separated by 12 to 15 amino acid residues, located close to the C terminus and a hydrophobic "motif 2" of 38 residues at the C terminus are the only essential and sufficient structural requirements for localizing a protein to the cell membrane. By these criteria, 73 genes are identified in the Giardia WB strain genome database as the putative repertoire of VSPs.

A minimal anaphase promoting complex/cyclosome (APC/C) in Trypanosoma brucei.

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 Trypanosoma brucei. 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.

Transition of a microRNA from repressing to activating translation depending on the extent of base pairing with the target.

MicroRNAs are major post-transcriptional regulators of gene expression. Here we show in the ancient protozoan Giardia lamblia a snoRNA-derived 26-nucleotide microRNA, miR3, which represses the translation of histone H2A mRNA containing an imperfect target but enhances translation when the target is made fully complementary. A stepwise mutational analysis of the fully complementary target showed that the activating effect of miR3 was significantly reduced when a single nucleotide at the 5'-end of the target was altered. The effect of miR3 became repressive when 12 of the nucleotides lost their complementation to miR3 with maximum repression reached when only 8 base-pairs remained between the miR3 seed sequence and the target. A synthetic 8-nucleotide RNA oligomer of the miR3 seed sequence was found capable of exerting a similar Argonaute-dependent translational repression. This is the first report showing a correlation between the extent of base-pairing with the target and a change in miRNA function.

Role of centrins 2 and 3 in organelle segregation and cytokinesis in Trypanosoma brucei.

Centrins are calcium binding proteins involved in cell division in eukaryotes. Previously, we have shown that depletion of centrin1 in Trypanosoma brucei (T. brucei) displayed arrested organelle segregation resulting in loss of cytokinesis. In this study we analyzed the role of T. brucei centrin2 (TbCen2) and T. brucei 3 (TbCen3) in the early events of T. brucei procyclic cell cycle. Both the immunofluorescence assay and electron microscopy showed that TbCen2 and 3-deficient cells were enlarged in size with duplicated basal bodies, multinuclei and new flagella that are detached along the length of the cell body. In both TbCen2 and TbCen3 depleted cells segregation of the organelles i.e. basal bodies, kinetoplast and nucleus was disrupted. Further analysis of the cells with defective organelle segregation identified three different sub configurations of organelle mis-segregations (Type 1-3). In addition, in majority of the TbCen2 depleted cells and in nearly half of the TbCen3 depleted cells, the kinetoplasts were enlarged and undivided. The abnormal segregations ultimately led to aborted cytokinesis and hence affected growth in these cells. Therefore, both centrin2 and 3 are involved in organelle segregation similar to centrin1 as was previously observed. In addition, we identified their role in kinetoplast division which may be also linked to overall mis-segregation.

The profile of snoRNA-derived microRNAs that regulate expression of variant surface proteins in Giardia lamblia.

In the current investigation, we analysed all the known small nucleolar RNAs (snoRNAs) in the deeply branching protozoan parasite Giardia lamblia for potential microRNAs (miRNAs) that might be derived from them. Two putative miRNAs have since been identified by Northern blot, primer extension, 3' RACE and co-immunoprecipitation with Giardia Argonaute (GlAgo), and designated miR6 and miR10. Giardia Dicer (GlDcr) is capable of processing the snoRNAs into the corresponding miRNAs in vitro. Potential miR6 and miR10 binding sites in Giardia genome were predicted bio-informatically. A miR6 binding site was found at the 3' untranslated regions (UTR) of 44 variant surface protein (vsp) genes, whereas a miR10 binding site was identified at the 3' end of 159 vsp open-reading frames. Thirty-three of these vsp genes turned out to contain binding sites for both miR6 and miR10. A reporter mRNA tagged with the 3' end of vsp1267, which contains the target sites for both miRNAs, was translationally repressed by both miRNAs in Giardia. Episomal expression of an N-terminal c-myc tagged VSP1267 was found significantly repressed by introducing either miR6 or miR10 into the cells and the repressive effects were additive. When the 2'-O-methyl antisense oligos (ASOs) of either miR6 or miR10 was introduced, however, there was an enhancement of tagged VSP1267 expression suggesting an inhibition of the repressive effects of endogenous miR6 or miR10 by the ASOs. Of the total 220 vsp genes in Giardia, we have now found 178 of them carrying putative binding sites for all the miRNAs that have been currently identified, suggesting that miRNAs are likely the regulators of VSP expression in Giardia.

The structural basis of localizing polo-like kinase to the flagellum attachment zone in Trypanosoma brucei.

The polo-like kinase in the deep branching eukaryote Trypanosoma brucei (TbPlk) has many unique features. Unlike all the other polo-like kinases known to associate with the nucleus and controlling both mitosis and cytokinesis, TbPlk localizes to the flagellum attachment zone (FAZ) and regulates only cytokinesis in T. brucei. TbPlk was, however, previously found capable of complementing all the multiple Plk (Cdc5) functions in Saccharomyces cerevisiae, indicating that it has acquired all the functions of Cdc5. In the present study, Cdc5 tagged with an enhanced yellow fluorescence protein (EYFP) localized exclusively in the FAZ of T. brucei, suggesting that the unusual localization and limited function of TbPlk are probably attributed to the particular environment in T. brucei cells. Structural basis for the FAZ localization of TbPlk was further investigated with TbPlk and TbPlk mutants tagged with EYFP and expressed in T. brucei. The results indicated that a kinase-inactive mutant N169A and a TbPlk mutant with the entire kinase domain (KD) deleted both localized to the FAZ. Substantial association with FAZ was also maintained when one of the two polo-boxes (PB1 or 2) or the linker region between them was deleted from TbPlk. But a deletion of both polo-boxes led to a complete exclusion of the protein from FAZ. All the deletion mutants retained the kinase activity, further indicating that the TbPlk kinase function does not play a role for FAZ localization. The two polo boxes in TbPlk are most likely instrumental in localizing the protein to FAZ through potential interactions with certain FAZ structural component(s). A putative cryptic bipartite nuclear targeting signal was identified in TbPlk, which was capable of directing TbPlk into the nucleus when either the kinase activity was lost or the PB1 was deleted from the protein.

A microRNA derived from an apparent canonical biogenesis pathway regulates variant surface protein gene expression in Giardia lamblia.

We have previously shown that a snoRNA-derived microRNA, miR2, in Giardia lamblia potentially regulates the expression of 22 variant surface protein (VSP) genes. Here, we identified another miRNA, miR4, also capable of regulating the expression of several VSPs but derived from an unannotated open reading frame (ORF) rather than a snoRNA, suggesting a canonical miRNA biogenesis pathway in Giardia. miR4 represses expression of a reporter containing two miR4 antisense sequences at the 3' UTR without causing a corresponding decrease in the mRNA level. This repression requires the presence of the Giardia Argonaute protein (GlAgo) and is reversed by 2' O-methylated antisense oligo to miR4, suggesting an RNA-induced silencing complex (RISC)-mediated mechanism. Furthermore, in vivo and in vitro evidence suggested that the Giardia Dicer protein (GlDcr) is required for miR4 biogenesis. Coimmunoprecipitation of miR4 with GlAgo further verified miR4 as a miRNA. A total of 361 potential target sites for miR4 were bioinformatically identified in Giardia, out of which 69 (32.7%) were associated with VSP genes. miR4 reduces the expression of a reporter containing two copies of the target site from VSP (GL50803_36493) at the 3' UTR. Sixteen of the 69 VSP genes were further found to contain partially overlapping miR2 and miR4 targeting sites. Expression of a reporter carrying the two overlapping sites was inhibited by either miR2 or miR4, but the inhibition was neither synergistic nor additive, suggesting a complex mechanism of miRNA regulation of VSP expression and the presence of a rich miRNAome in Giardia.

Gene regulation in Giardia lambia involves a putative microRNA derived from a small nucleolar RNA.

Two core microRNA (miRNA) pathway proteins, Dicer and Argonaute, are found in Giardia lamblia, a deeply branching parasitic protozoan. There are, however, no apparent homologues of Drosha or Exportin5 in the genome. Here, we report a 26 nucleotide (nt) RNA derived from a 106 nt Box C/D snoRNA, GlsR2. This small RNA, designated miR5, localizes to the 3' end of GlsR2 and has a 75 nt hairpin precursor. GlsR2 is processed by the Dicer from Giardia (GlDcr) and generated miR5. Immunoprecipitation of the Argonaute from Giardia (GlAgo) brought down miR5. When a Renilla Luciferase transcript with a 26 nt miR5 antisense sequence at the 3'-untranslated region (3' UTR) was introduced into Giardia trophozoites, Luciferase expression was reduced ∼25% when synthetic miR5 was also introduced. The Luciferase mRNA level remained, however, unchanged, suggesting translation repression by miR5. This inhibition was fully reversed by introducing also a 2'-O-methylated antisense inhibitor of miR5, suggesting that miR5 acts by interacting specifically with the antisense sequence in the mRNA. A partial antisense knock down of GlDcr or GlAgo in Giardia indicated that the former is needed for miR5 biogenesis whereas the latter is required for miR5-mediated translational repression. Potential targets for miR5 with canonical seed sequences were predicted bioinformatically near the stop codon of Giardia mRNAs. Four out of the 21 most likely targets were tested in the Luciferase reporter assay. miR5 was found to inhibit Luciferase expression (∼20%) of transcripts carrying these potential target sites, indicating that snoRNA-derived miRNA can regulate the expression of multiple genes in Giardia.

A La autoantigen homologue is required for the internal ribosome entry site mediated translation of giardiavirus.

Translation of Giardiavirus (GLV) mRNA is initiated at an internal ribosome entry site (IRES) in the viral transcript. The IRES localizes to a downstream portion of 5' untranslated region (UTR) and a part of the early downstream coding region of the transcript. Recent studies indicated that the IRES does not require a pre-initiation complex to initiate translation but may directly recruit the small ribosome subunit with the help of a number of trans-activating protein factors. A La autoantigen homologue in the viral host Giardia lamblia, GlLa, was proposed as one of the potential trans-activating factors based on its specific binding to GLV-IRES in vitro. In this study, we further elucidated the functional role of GlLa in GLV-IRES mediated translation in Giardia by knocking down GlLa with antisense morpholino oligo, which resulted in a reduction of GLV-IRES activity by 40%. An over-expression of GlLa in Giardia moderately stimulated GLV-IRES activity by 20%. A yeast inhibitory RNA (IRNA), known to bind mammalian and yeast La autoantigen and inhibit Poliovirus and Hepatitis C virus IRES activities in vitro and in vivo, was also found to bind to GlLa protein in vitro and inhibited GLV-IRES function in vivo. The C-terminal domain of La autoantigen interferes with the dimerization of La and inhibits its function. An over-expression of the C-terminal domain (200-348aa) of GlLa in Giardia showed a dominant-negative effect on GLV-IRES activity, suggesting a potential inhibition of GlLa dimerization. HA tagged GlLa protein was detected mainly in the cytoplasm of Giardia, thus supporting a primary role of GlLa in translation initiation in Giardiavirus.

Rab11 and actin cytoskeleton participate in Giardia lamblia encystation, guiding the specific vesicles to the cyst wall.

BACKGROUND: Giardia passes through two stages during its life cycle, the trophozoite and the cyst. Cyst formation involves the synthesis of cyst wall proteins (CWPs) and the transport of CWPs into encystation-specific vesicles (ESVs). Active vesicular trafficking is essential for encystation, but the molecular machinery driving vesicular trafficking remains unknown. The Rab proteins are involved in the targeting of vesicles to several intracellular compartments through their association with cytoskeletal motor proteins. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we found a relationship between Rab11 and the actin cytoskeleton in CWP1 transport. Confocal microscopy showed Rab11 was distributed throughout the entire trophozoite, while in cysts it was translocated to the periphery of the cell, where it colocalized with ESVs and microfilaments. Encystation was also accompanied by changes in rab11 mRNA expression. To evaluate the role of microfilaments in encystation, the cells were treated with latrunculin A. Scanning electron microscopy showed this treatment resulted in morphological damages to encysted parasites. The intensity of fluorescence-labeled Rab11 and CWP1 in ESVs and cyst walls was reduced, and rab11 and cwp1 mRNA levels were down-regulated. Furthermore, knocking down Rab11 with a hammerhead ribozyme resulted in an up to 80% down-regulation of rab11 mRNA. Although this knockdown did not appear lethal for trophozoites and did not affect cwp1 expression during the encystation, confocal images showed CWP1 was redistributed throughout the cytosol. CONCLUSIONS AND SIGNIFICANCE: Our results indicate that Rab11 participates in the early and late encystation stages by regulating CWP1 localization and the actin-mediated transport of ESVs towards the periphery. In addition, alterations in the dynamics of actin affected rab11 and cwp1 expression. Our results provide new information about the molecules involved in Giardia encystation and suggest that Rab11 and actin may be useful as novel pharmacological targets.

Polo-like kinase guides cytokinesis in Trypanosoma brucei through an indirect means.

Polo-like kinase in Trypanosoma brucei (TbPLK) is confined to the flagellum attachment zone (FAZ) and regulates only cytokinetic initiation. However, it apparently diffuses into the cytoplasm before the trans-localization of chromosomal passenger complex (CPC) from the midzone of central spindle to FAZ, which is known to be required for initiating cytokinesis. Synchronized T. brucei procyclic cells treated with a TbPLK inhibitor, GW843682X (GW), in late S phase were found to go through a full cell cycle at a normal pace before being arrested at cytokinetic initiation in the second cycle. However, synchronized cells treated with GW in G(1) phase were arrested at cytokinetic initiation within the first cell cycle, suggesting that inhibition of TbPLK at its emergence blocks cytokinesis within the same cell cycle. To rule out potential off-target effects from GW, TbPLK RNA interference (RNAi) was induced to deplete TbPLK, and the progression of synchronized cells from late S phase was also found to be arrested at cytokinetic initiation within the first cell cycle. Apparently, TbPLK has accomplished its role in guiding cytokinesis before the late S phase, presumably by phosphorylating a certain substrate(s) during S phase, which may play a critical role in initiating the subsequent cytokinesis.

Giardiavirus internal ribosome entry site has an apparently unique mechanism of initiating translation.

Giardiavirus (GLV) utilizes an internal ribosome entry site (IRES) for translation initiation in the early branching eukaryote Giardia lamblia. Unlike most of the viral IRESs among higher eukaryotes, which localize primarily within the 5'-untranslated region (UTR), the GLV IRES comprises 253 nts of 5'UTR and the initial 264 nts in the open-reading-frame (ORF). To test if GLV IRES also functions in higher eukaryotic systems, we examined it in rabbit reticulocyte lysate (RRL) and found that it functions much less efficiently than the IRES from the Encephalomyocarditis virus (EMCV) or Cricket paralysis virus (CrPV). In contrast, both EMCV-IRES and CrPV-IRESs were inactive in transfected Giardia cells. Structure-function analysis indicated that only the stem-loop U5 from the 5'UTR and the stem-loop I plus the downstream box (Dbox) from the ORF of GLV IRES are required for limited IRES function in RRL. Edeine, a translation initiation inhibitor, did not significantly affect the function of GLV IRES in either RRL or Giardia, indicating that a pre-initiation complex is not required for GLV IRES-mediated translation initiation. However, the small ribosomal subunit purified from Giardia did not bind to GLV IRES, indicating that additional protein factors may be necessary. A member of the helicase family IBP1 and two known viral IRES binding proteins La autoantigen and SRp20 have been identified in Giardia that bind to GLV IRES in vitro. These three proteins could be involved in facilitating small ribosome recruitment for initiating translation.

CRK9 contributes to regulation of mitosis and cytokinesis in the procyclic form of Trypanosoma brucei.

BACKGROUND: The Trypanosoma brucei cell cycle is regulated by combinations of cyclin/CRKs (cdc2 related kinases). Recently, two additional cyclins (CYC10, CYC11) and six new CRK (CRK7-12) homologues were identified in the T. brucei genome database 12. RESULTS: Individual RNAi knockdowns of these new proteins in the procyclic form of T. brucei showed no apparent phenotype except for the CRK9 depletion, which enriched the cells in G2/M phase. But a similar CRK9 knockdown in the bloodstream form caused no apparent phenotype. CRK9 lacks the typical PSTAIRE motif for cyclin binding and the phenylalanine "gatekeeper" but binds to cyclin B2 in vitro and localizes to the nucleus in both forms of T. brucei. CRK9-depleted procyclic-form generated no detectable anucleate cells, suggesting an inhibition of cytokinesis by CRK9 depletion as well. The knockdown enriched cells with one nucleus, one kinetoplast and two closely associated basal bodies with an average distance of 1.08 mm in between, which was shorter than the control value of 1.36 microm, and the cells became morphologically deformed and rounded with time. CONCLUSION: CRK9 may play a role in mediating the segregation between the two kinetoplast/basal body pairs prior to cytokinetic initiation. Since such a segregation over a relatively significant distance is essential for cytokinetic initiation only in the procyclic but may not be in the bloodstream form, CRK9 could be specifically involved in regulating cytokinetic initiation in the procyclic form of T. brucei.

snoRNA, a novel precursor of microRNA in Giardia lamblia.

An Argonaute homolog and a functional Dicer have been identified in the ancient eukaryote Giardia lamblia, which apparently lacks the ability to perform RNA interference (RNAi). The Giardia Argonaute plays an essential role in growth and is capable of binding specifically to the m(7)G-cap, suggesting a potential involvement in microRNA (miRNA)-mediated translational repression. To test such a possibility, small RNAs were isolated from Giardia trophozoites, cloned, and sequenced. A 26-nucleotide (nt) small RNA (miR2) was identified as a product of Dicer-processed snoRNA GlsR17 and localized to the cytoplasm by fluorescence in situ hybridization, whereas GlsR17 was found primarily in the nucleolus of only one of the two nuclei in Giardia. Three other small RNAs were also identified as products of snoRNAs, suggesting that the latter could be novel precursors of miRNAs in Giardia. Putative miR2 target sites were identified at the 3'-untranslated regions (UTR) of 22 variant surface protein mRNAs using the miRanda program. In vivo expression of Renilla luciferase mRNA containing six identical miR2 target sites in the 3'-UTR was reduced by 40% when co-transfected with synthetic miR2, while the level of luciferase mRNA remained unaffected. Thus, miR2 likely affects translation but not mRNA stability. This repression, however, was not observed when Argonaute was knocked down in Giardia using a ribozyme-antisense RNA. Instead, an enhancement of luciferase expression was observed, suggesting a loss of endogenous miR2-mediated repression when this protein is depleted. Additionally, the level of miR2 was significantly reduced when Dicer was knocked down. In all, the evidence indicates the presence of a snoRNA-derived miRNA-mediated translational repression in Giardia.

The chromosomal passenger complex and a mitotic kinesin interact with the Tousled-like kinase in trypanosomes to regulate mitosis and cytokinesis.

Aurora B kinase plays essential roles in mitosis and cytokinesis in eukaryotes. In the procyclic form of Trypanosoma brucei, the Aurora B homolog TbAUK1 regulates mitosis and cytokinesis, phosphorylates the Tousled-like kinase TbTLK1, interacts with two mitotic kinesins TbKIN-A and TbKIN-B and forms a novel chromosomal passenger complex (CPC) with two novel proteins TbCPC1 and TbCPC2. Here we show with time-lapse video microscopy the time course of CPC trans-localization from the spindle midzone in late anaphase to the dorsal side of the cell where the anterior end of daughter cell is tethered, and followed by a glide toward the posterior end to divide the cell, representing a novel mode of cytokinesis in eukaryotes. The three subunits of CPC, TbKIN-B and TbTLK1 interact with one another suggesting a close association among the five proteins. An ablation of TbTLK1 inhibited the subsequent trans-localization of CPC and TbKIN-B, whereas a knockdown of CPC or TbKIN-B disrupted the spindle pole localization of TbTLK1 during mitosis. In the bloodstream form of T. brucei, the five proteins also play essential roles in chromosome segregation and cytokinesis and display subcellular localization patterns similar to that in the procyclic form. The CPC in bloodstream form also undergoes a trans-localization during cytokinesis similar to that in the procyclic form. All together, our results indicate that the five-protein complex CPC-TbTLK1-TbKIN-B plays key roles in regulating chromosome segregation in the early phase of mitosis and that the highly unusual mode of cytokinesis mediated by CPC occurs in both forms of trypanosomes.

KMP-11, a basal body and flagellar protein, is required for cell division in Trypanosoma brucei.

Kinetoplastid membrane protein 11 (KMP-11) has been identified as a flagellar protein and is conserved among kinetoplastid parasites, but its potential function remains unknown. In a recent study, we identified KMP-11 as a microtubule-bound protein localizing to the flagellum as well as the basal body in both procyclic and bloodstream forms of Trypanosoma brucei (Z. Li, J. H. Lee, F. Chu, A. L. Burlingame, A. Gunzl, and C. C. Wang, PLoS One 3:e2354, 2008). Silencing of KMP-11 by RNA interference inhibited basal body segregation and cytokinesis in both forms and resulted in multiple nuclei of various sizes, indicating a continuous, albeit somewhat defective, nuclear division while cell division was blocked. KMP-11 knockdown in the procyclic form led to severely compromised formation of the new flagellum attachment zone (FAZ) and detachment of the newly synthesized flagellum. However, a similar phenotype was not observed in the bloodstream form depleted of KMP-11. Thus, KMP-11 is a flagellar protein playing critical roles in regulating cytokinesis in both forms of the trypanosomes. Its distinct roles in regulating FAZ formation in the two forms may provide a clue to the different mechanisms of cytokinetic initiation in procyclic and bloodstream trypanosomes.

Polo-like kinase is expressed in S/G2/M phase and associated with the flagellum attachment zone in both procyclic and bloodstream forms of Trypanosoma brucei.

Trypanosoma brucei, the etiologic agent of African sleeping sickness, divides into insect (procyclic) and bloodstream forms. These two forms are subject to distinct cell cycle regulations, with cytokinesis controlled primarily by basal body/kinetoplast segregation in the procyclic form but by mitosis in the bloodstream form. Polo-like kinases (PLKs), known to play essential roles in regulating both mitosis and cytokinesis among eukaryotes, have a homologue in T. brucei, TbPLK, which regulates only cytokinesis. In our previous study, overexpressed triply hemagglutinin-tagged TbPLK (TbPLK-3HA) in the procyclic form localized to a mid-dorsal point and the anterior tip of the cell along the flagellum attachment zone (FAZ). In our current study, TbPLK-3HA expressed at the endogenous level was identified at the same dorsal location of both procyclic and bloodstream forms, albeit it was no longer detectable at the anterior tip of the cell. Endogenously expressed TbPLK fused with an enhanced yellow fluorescent protein (EYFP) localized to the same dorsal location along the FAZs in living procyclic and bloodstream cells. Fluorescence-activated cell sorter analysis of hydroxyurea-synchronized procyclic cells revealed that TbPLK-EYFP emerges during S phase, persists through G(2)/M phase, and vanishes in G(1) phase. An indicated TbPLK-EYFP association with the FAZs of G(2)/M cells may thus represent a timely localization to a potential initiation site of cytokinesis, which agrees with the recognized role of TbPLK in cytokinetic initiation.