TbAGO1, an Argonaute protein required for RNA interference, is involved in mitosis and chromosome segregation in Trypanosoma brucei

Argonaute proteins (AGOs) are central to RNA interference (RNAi) and related silencing pathways. At the core of the RNAi pathway in the ancient parasitic eukaryote Trypanosoma brucei is a single Argonaute protein, TbAGO1, with an established role in the destruction of potentially harmful retroposon transcripts. One notable feature of TbAGO1 is that a fraction sediments with polyribosomes, and this association is facilitated by an arginine/glycine-rich domain (RGG domain) at the N terminus of the protein. Here we report that reducing the size of the RGG domain and, in particular, mutating all arginine residues severely reduced the association of TbAGO1 with polyribosomes and RNAi-induced cleavage of mRNA. However, these mutations did not change the cellular localization of Argonaute and did not affect the accumulation of single-stranded siRNAs, an essential step in the activation of the RNA-induced silencing complex. We further show that mRNA on polyribosomes can be targeted for degradation, although this alliance is not a pre-requisite. Finally, sequestering tubulin mRNAs from translation with antisense morpholino oligonucleotides reduced the RNAi response indicating that mRNAs not engaged in translation may be less accessible to the RNAi machinery. We conclude that the association of the RNAi machinery and target mRNA on polyribosomes promotes an efficient RNAi response. This mechanism may represent an ancient adaptation to ensure that retroposon transcripts are efficiently destroyed, if they become associated with the translational apparatus.

Section: The Size Of the Tbago1 Rgg Domain And The Presence Of Arginimentioning

confidence: 99%

RNA Interference in Trypanosoma brucei

Shi

Chamond

Djikeng

et al. 2009

“…Functional genomic studies in T. brucei have also shown that non-mitotic proteins could play a role in mitotic progression. For example, knockdown of TbAGO1, which itself is required for RNAi, produced abnormal mitotic phenotypes (10).…”

mentioning

confidence: 99%

An Essential Cell Cycle-regulated Nucleolar Protein Relocates to the Mitotic Spindle Where It Is Involved in Mitotic Progression in Trypanosoma brucei

Boucher¹,

Dacheux²,

Giroud³

et al. 2007

Trypanosoma brucei is an ancient protozoan parasite and an agent of important diseases in human (African sleeping sickness) and cattle (nagana). The cell division cycle in T. brucei displays unique characteristics compared with other eukaryotes (1), and cellular morphology is determinant in the cell cycle progression (2). Indeed, cell division requires replication and segregation of the nucleus, kinetoplast (representing the mitochondrial DNA), flagellum, and basal body. The sequential G 1 , S, G 2 , and M phases that constitute the eukaryotic nuclear cell cycle is present in trypanosomes (3). Briefly, G 1 cells have one nucleus and one kinetoplast (1N1K). The kinetoplast and nuclear DNA have a discrete S phase such that segregation of the replicated kinetoplast is completed before onset of mitosis. This results in 1N2K cells in late G 2 , followed by mitosis (M phase) resulting in 2N2K cells and then cytokinesis producing two 1N1K cells.Mitosis must ensure faithful transmission of the nuclear genetic material to daughter cells. A more detailed model of the G 2 /M transition is starting to emerge in trypanosomes, a model that contrasts with the general eukaryotic model wherein numerous cellular checkpoints tightly coordinate mitotic progression and cytokinesis. Compared with other eukaryotes, cell cycle regulation in T. brucei appears simpler, as only a limited number of conserved mitotic proteins involved in the G 2 /M phase have been identified: cyclin homologs (CYC6 (4) and CycB2 (5)), cdc2-related kinase (CRK3) (6), aurora kinase homologue (TbAUK1) (7) and homologues of the anaphasepromoting complex/cyclosome (APC1 and CDC27) (8). Different cell cycle mechanisms operate as the parasite alternates between the procyclic (insect stage) and bloodstream form (mammalian stage). In procyclic cells, there is a dissociation of mitosis and cytokinesis such that entry into cytokinesis does not rely on mitosis or nuclear synthesis but mainly on basal body segregation (9). Inhibition of mitosis by RNAi 2 (CYC6 (4) and CRK3 (6)) or with anti-microtubule agents (2) results in the generation of anucleated cells (zoids) (0N1K) and cells with an enlarged nucleus (1N*1K, 1N* being a replicated DNA delayed in mitosis). This suggests novel cell cycle checkpoints and contrasts with bloodstream cells, which cannot enter cytokinesis under the same conditions (6). Accordingly, the study of mitotic proteins in T. brucei has highlighted distinctive stagespecific phenotypes following mitotic disorders. Depletion of the mitotic cyclin CYC6 caused enrichment of 0N1K in procyclic forms and cells with one nucleus and multiple kinetoplasts

“…However, in contrast to other organisms, only one RNAi component has so far been identified in the parasite. The polypeptide is a member of the AGO protein family and has been termed TbAGO1 (38,39). TbAGO1 is essential for RNAi in insect stage trypanosomes and was shown to be assembled in siRNA-associated ribonucleoprotein particles, which are important for the stabilization and/or generation of siRNAs (39).…”

mentioning

confidence: 99%

In Vitro Synthesized Small Interfering RNAs Elicit RNA Interference in African Trypanosomes

Best

Handoko²,

Schlüter

et al. 2005

RNA interference (RNAi) describes an epigenetic gene silencing reaction by which gene-specific doublestranded RNA acts as a trigger to induce the ribonucleolytic degradation of homologous transcripts. RNAi in African trypanosomes has been shown to be involved in regulating the transcript abundance of retroposons, and the process currently represents the method of choice in gene function studies of the parasite. However, little is known concerning the mechanistic and structural aspects of the processing reaction. This is in part due to the absence of a trypanosome-specific RNAi in vitro system. Here we demonstrate that both the Dicer and the RNA-induced silencing complex steps of the RNAi reaction pathway can be monitored in vitro using cell-free trypanosome extracts. The two in vitro activities and the generated small interfering RNAs (siRNAs) are characterized by features known from other organisms, and we demonstrate that chemically as well as enzymatically synthesized siRNAs are functional in the parasite. Thus, the transfection of synthetic siRNAs can be used to rapidly monitor gene knockdown phenotypes in Trypanosoma brucei, which should be helpful in genome-wide, RNAi-based screening experiments.