Durand-Dubief Mickaël, Absalon Sabrina, Menzer Linda, Ngwabyt Sandra, Ersfeld Klaus, Bastin Philippe
Régulation et Dynamique des Génomes, Muséum National d'Histoire Naturelle, INSERM & CNRS, 75005 Paris, France.
Mol Biochem Parasitol. 2007 Dec;156(2):144-53. doi: 10.1016/j.molbiopara.2007.07.016. Epub 2007 Aug 1.
The protist Trypanosoma brucei possesses a single Argonaute gene called TbAGO1 that is necessary for RNAi silencing. We previously showed that in strain 427, TbAGO1 knock-out leads to a slow growth phenotype and to chromosome segregation defects. Here we report that the slow growth phenotype is linked to defects in segregation of both large and mini-chromosome populations, with large chromosomes being the most affected. These phenotypes are completely reversed upon inducible re-expression of TbAGO1 fused to GFP, demonstrating their link with TbAGO1. Trypanosomes that do not express TbAGO1 show a general increase in the abundance of transcripts derived from the short retroposon RIME (Ribosomal Interspersed Mobile Element). Supplementary large RIME transcripts emerge in the absence of RNAi, a phenomenon coupled to the disappearance of short transcripts. These fluctuations are reversed by inducible expression of GFP::TbAGO1. Furthermore, we use a combination of Northern blots, RT-PCR and sequencing to reveal that RNAi controls expression of transcripts derived from RHS (Retrotransposon Hot Spot) pseudogenes (RHS genes with retro-element(s) integrated within their coding sequence). Absence of RNAi also leads to an increase of steady-state transcripts from regular RHS genes (those without retro-element), indicating a role for pseudogene in control of gene expression. However, analysis of retroposon abundance and arrangement in the genome of multiple clonal cell lines of TbAGO1-/- failed to reveal movement of mobile elements despite the increased amounts of retroposon transcripts.
原生生物布氏锥虫拥有一个名为TbAGO1的单一AGO蛋白基因，该基因对于RNA干扰沉默是必需的。我们之前表明，在427菌株中，TbAGO1基因敲除会导致生长缓慢的表型以及染色体分离缺陷。在此我们报告，生长缓慢的表型与大染色体和小染色体群体分离的缺陷有关，其中大染色体受影响最大。当可诱导地重新表达与绿色荧光蛋白（GFP）融合的TbAGO1时，这些表型完全逆转，证明了它们与TbAGO1的联系。不表达TbAGO1的锥虫显示出源自短反转录转座子RIME（核糖体散布移动元件）的转录本丰度普遍增加。在没有RNA干扰的情况下会出现额外的大RIME转录本，这一现象与短转录本的消失相关。这些波动通过可诱导表达GFP::TbAGO1而逆转。此外，我们结合使用Northern印迹、逆转录聚合酶链反应（RT-PCR）和测序来揭示RNA干扰控制源自RHS（反转录转座子热点）假基因（在其编码序列内整合了反转录元件的RHS基因）的转录本表达。缺乏RNA干扰也会导致来自常规RHS基因（那些没有反转录元件的基因）的稳态转录本增加，表明假基因在基因表达控制中起作用。然而，对TbAGO1基因敲除的多个克隆细胞系基因组中的反转录转座子丰度和排列进行分析，尽管反转录转座子转录本数量增加，但未能揭示移动元件的移动情况。
