Argonaute Protein in the Early Divergent Eukaryote Trypanosoma brucei: Control of Small Interfering RNA Accumulation and Retroposon Transcript Abundance

Abstract

Members of the Argonaute protein family have been linked through a combination of genetic and biochemical studies to RNA interference (RNAi) and related phenomena. Here, we describe the characterization of the first Argonaute protein (AGO1) in Trypanosoma brucei, the earliest divergent eukaryote where RNAi has been described so far. AGO1 is predominantly cytoplasmic and is found in a ribonucleoprotein particle with small interfering RNAs (siRNAs), and this particle is present in a soluble form, as well as associated with polyribosomes. A genetic knockout of AGO1 leads to a loss of RNAi, and concomitantly, endogenous retroposonderived siRNAs as well as siRNAs derived from transgenic double-stranded RNA are reduced to almost undetectable levels. Furthermore, AGO1 deficiency leads to an increase in retroposon transcript abundance via mechanisms operating at the transcriptional level and at the RNA stability level. Our results suggest that AGO1 function is required for production and/or stabilization of siRNAs and provide the first evidence for an Argonaute protein being involved in the regulation of retroposon transcript levels.

Expression site silencing and life-cycle progression appear normal in Argonaute1-deficient Trypanosoma brucei

“…1C), whereas the accumulation of INGI siRNAs was significantly reduced (Fig. 1D), similar to the results obtained for procyclic trypanosomes [21,24].…”

“…1C), whereas the accumulation of INGI siRNAs was significantly reduced (Fig. 1D), similar to the results obtained for procyclic trypanosomes [21,24].We then examined whether the loss of AGO1 affected monoallelic VSG expression, by comparing the steady-state levels of several VSG RNAs in Lister 427 wild-type and ago1 −/− trypanosomes, by RT-PCR. We chose specific primer pairs to detect transcripts of VSG 221 (single-copy gene, active ES), VSG bR2 (2 telomeric and 1 internal copy), VSG 118 (singlecopy gene, inactive ES), VSG VO 2 (1 telomeric and 1 internal copy) or VSG B (1 telomeric and 2 internal copies).…”

On the origin and functions of RNA-mediated silencing: from protists to man

“…Arabidopsis Ago4 has been implicated in DNA and histone methylation whereas Ago1 is an RNA slicer and much more pleotropic in its functions (Baumberger and Baulcombe 2005;Matzke and Birchler 2005;Qi et al 2005;Zilberman et al 2003). Interestingly, in organisms that encode a single Ago-Piwi protein, such as S. pombe and T. brucei, its mutation results in defects in both transcriptional and post-transcriptional silencing (Shi et al 2004;Sigova et al 2004). …”

“…RNAi-dependent transcriptional silencing has been demonstrated to entail histone modifications, such as H3K9 methylation, in eukaryotes belonging to at least three different supergroups (Table 2). Moreover, siRNA-triggered transcriptional repression occurs in organisms that lack cytosine DNA methylation such as S. pombe and C. elegans (Grishok et al 2005;Martienssen et al 2005;Ponger and Li 2005;Robert et al 2005;Volpe et al 2002) and in organisms with very limited DNA methylation such as T. brucei and D. melanogaster (Kavi et al 2005;Pal-Bhadra et al 2004;Ponger and Li 2005;Shi et al 2004;Ullu et al 2004). In contrast, RNA-directed DNA methylation has, thus far, only been demonstrated in plants and mammals (Kawasaki and Taira 2004;Matzke and Birchler 2005;Morris et al 2004) and the role of DNA methylation in this type of gene silencing is somewhat debatable in mammals (Ting et al 2005;Weinberg et al 2006).…”

A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe

“…siRNAs are assembled into a protein-RNA complex, the RNAinduced silencing complex (RISC), which directs cleavage of the target RNA (Hammond et al 2000;Zamore et al 2000;Nykänen et al 2001). Among the various protein factors required for RNAi, members of the Argonaute family of proteins are universally associated with siRNA-directed gene silencing (Cogoni and Macino 1997;Tabara et al 1999Tabara et al , 2002Cerutti et al 2000;Fagard et al 2000;Catalanotto et al 2002;Kennerdell et al 2002;Mochizuki et al 2002;Pal-Bhadra et al 2002;Tijsterman et al 2002;Williams and Rubin 2002;Shi et al 2004). Plants and animals contain multiple Argonaute paralogs; the Drosophila genome encodes at least five distinct Argonaute proteins; humans have eight; and worms, 27!…”