Regulation of Trypanosoma brucei Cell Cycle

We have recently dissected the cell cycle of Trypanosoma brucei, the causative pathogen of African sleeping sickness, and made several interesting discoveries; (1) the cell division of T. brucei is regulated primarily by mitosis in the bloodstream form but by the replication of the single mitochondrion  in the insect form; (2) the chromosomal passenger complex (CPC) in T. brucei consists of Aurora kinase B and two other novel proteins.  It trans-localizes from the midzone of the central spindle in the nucleus to the flagellar attachment zone (FAZ) on the cell membrane during telophase and moves along the FAZ to the anterior end of the dividing cell.  It then glides all the way from the anterior to the posterior end in an action like opening a zipper to divide the cell into two.  It represents a novel mechanism of cytokinesis never observed in any other eukaryotes previously; (3) the polo-like kinase of T. brucei (TbPlk) has all the multiple functions in regulating mitosis and cytokinesis when expressed in yeast.  But it only localizes to the FAZ and regulates cytokinesis in T. brucei presumably due to the unique cellular environment in the latter.  We are currently in the process of identifying the protein factor(s) involved in localizing TbPlk to the FAZ and mediating the trans-localization of CPC to the FAZ to initiate cytokinesis in T. brucei.  This highly distinctive mode of cell division will be not only of great significance in helping our understanding of the evolution of cell division but also in opening up a good opportunity for chemotherapy against this pathogen.

We also found that the anaphase promoting complex (APC) in T. brucei consists of only a very limited number of subunit proteins.  No homologues of the subunits of the spindle assembly checkpoint complex could be identified in the genomic database of T. brucei.  In order to understand how the critical metaphase to anaphase transition in T. brucei is being regulated, we performed TAP-tagging of the few identified APC subunits, isolated the protein complexes during the synchronized metaphase and anaphase and compared them in proteomic analysis.  A few novel proteins have since been identified, which could lead to isolation of a distinct machinery controlling the metaphase-anaphase transition in this deeply branching eukaryote with high interest in evolutionary biology and great potential for anti-trypanosomiasis chemotherapy.

Regulation of Gene Expression in Giardia lamblia

Giardia is a deeply branching protozoan and one of the most ancient eukaryotes known to man.  It is widely distributed and causes diarrhea in man; commonly known as giardiasis.  One of the unusual features of this organism is in the very short 5’-UTRs and 3’-UTRs in its mRNAs.  The mechanism of regulating gene expression in Giardia is virtually unknown.  We recently identified in this organism a 26 nucleotide small RNA that is derived from the 3’-end of a snoRNA, GlsR17.  It represses the translation of a reporter transcript carrying an antisense sequence at the 3’-UTR in Giardia.  The action depends on the presence of an Argonaute homologue and the maturation of the small RNA requires the action of a Dicer homologue in Giardia.  Bioinformatic analysis identified many mRNAs with potential binding sites at the 3’-ends for the small RNA.  A few of them were tested as the 3’-UTR of a reporter transcript and found to mediate translation repression by the small RNA.  This small RNA, designated microRNA2 (miR2), is among the first microRNAs derived from a snoRNA and the first identified in Giardia.

Among the mRNAs carrying the potential binding sites for miR2, 22 are mRNAs encoding variant surface proteins (VSP).  Three additional snoRNA-derived microRNAs (miR5, miR6 and miR10) and another microRNA from a hypothetical open reading frame, miR4, have since been discovered and thoroughly characterized and all found to play a regulatory role on the expression of VSPs in Giardia.  Essentially, all the ~200 VSP genes in Giardia have been found to carry at least one or more of the potential binding sites for the five microRNAs.  Though more microRNAs are likely involved in this regulatory mechanism, it is likely that a microRNA machinery may function as the primary regulatory of VSP expression in this ancient eukaryote.

By immunoprecipitating the Argonaute homologue from Giardia, we were able to bring down a discrete 26-28 nucleotide small RNA band corresponding to the size of the microRNAs in this organism.  The purified small RNAs were converted to cDNAs and sequenced.  Among the thousands of individual sequences thus obtained, the five known microRNA sequences were found, but most of the other microRNA sequences identified in metazoa thus far were missing from the library, suggesting that the microRNA machinery in Giardia is separated from those in metazoa by a long evolutionary distance.
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