Trypanosoma brucei PUF9 regulates mRNAs for proteins involved in replicative processes over the cell cycle.
Trypanosoma brucei PUF9 regulates mRNAs for proteins involved in replicative processes over the cell cycle.
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DOI：
10.1371/journal.ppat.1000565
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发表时间：
2009-08
期刊：
影响因子：
6.7
通讯作者：
Clayton C
中科院分区：
文献类型：
--
作者：
Archer SK;Luu VD;de Queiroz RA;Brems S;Clayton C
在细胞周期中的特定点所需的许多基因表现出细胞周期依赖性表达。在早期分化模型真核生物和重要的人类病原体布氏锥虫中，细胞周期和其他过程中的基因表达调控几乎完全是转录后的。在这里，我们表明，T。布鲁氏菌RNA结合蛋白PUF 9在S期期间稳定某些转录物。PUF 9的靶转录物-LIGKA、PNT 1和PNT 2-通过用TAP标记的PUF 9的亲和纯化来鉴定。针对PUF 9的RNAi引起细胞在G2/M期的积累，并且出乎意料地使PUF 9靶mRNA不稳定，尽管大多数已知的Puf结构域蛋白促进其靶mRNA的降解。PUF 9调节的转录物的水平是细胞周期依赖性的，在S期中期至晚期达到峰值，并且当PUF 9被RNAi靶向时，这种作用被消除。序列UUGUACC在PUF 9靶标的3′ UTR中过度表达;该基序中的点突变消除了携带PNT 1 3′ UTR的报告转录本的PUF 9依赖性稳定性。LIGKA参与复制的动基体，在这里，我们表明，PNT 1也是动基体相关的，其过度表达导致动基体相关的缺陷，而PNT 2是本地化的细胞核在G1期和有丝分裂过程中重新分配到有丝分裂纺锤体。PUF 9靶点可能构成转录后调节子，编码参与G2早期时间协调复制过程的蛋白质。单细胞原生动物布氏锥虫是非洲昏睡病的病原体，每年造成10万多人死亡，并与其他重要病原体（如利什曼原虫和克氏锥虫）有关。不寻常的是，这些生物体并不是通过改变它们复制成RNA的速率来调节它们的基因，而是通过改变RNA破坏的速率或翻译成蛋白质的速率。我们发现了一种RNA结合蛋白PUF 9，它负责在细胞分裂周期的特定时间点（DNA复制后）积累几种RNA分子。相应地，由这些RNA编码的蛋白质似乎在这个时间点或之后不久在各种细胞结构的分裂中起作用。其中两个促进动基体（一种含有线粒体DNA的细胞器）的复制，而另一个在有丝分裂纺锤体中被发现。它们的时间共表达可能源于锥虫的另一个不寻常的特征：每个细胞只存在一个动质体（和其他几个细胞器），它们的复制与细胞分裂协调。事实上，PUF 9在控制细胞器拷贝数方面可能很重要，因为PUF 9的抑制导致细胞具有太多的动质体、鞭毛或细胞核。
Many genes that are required at specific points in the cell cycle exhibit cell cycle–dependent expression. In the early-diverging model eukaryote and important human pathogen Trypanosoma brucei, regulation of gene expression in the cell cycle and other processes is almost entirely post-transcriptional. Here, we show that the T. brucei RNA-binding protein PUF9 stabilizes certain transcripts during S-phase. Target transcripts of PUF9—LIGKA, PNT1 and PNT2—were identified by affinity purification with TAP-tagged PUF9. RNAi against PUF9 caused an accumulation of cells in G2/M phase and unexpectedly destabilized the PUF9 target mRNAs, despite the fact that most known Puf-domain proteins promote degradation of their target mRNAs. The levels of the PUF9-regulated transcripts were cell cycle dependent, peaking in mid- to late- S-phase, and this effect was abolished when PUF9 was targeted by RNAi. The sequence UUGUACC was over-represented in the 3′ UTRs of PUF9 targets; a point mutation in this motif abolished PUF9-dependent stabilization of a reporter transcript carrying the PNT1 3′ UTR. LIGKA is involved in replication of the kinetoplast, and here we show that PNT1 is also kinetoplast-associated and its over-expression causes kinetoplast-related defects, while PNT2 is localized to the nucleus in G1 phase and redistributes to the mitotic spindle during mitosis. PUF9 targets may constitute a post-transcriptional regulon, encoding proteins involved in temporally coordinated replicative processes in early G2 phase. The unicellular protozoan Trypanosoma brucei is the causative agent of African sleeping sickness, responsible for over 100,000 deaths annually, and is related to other important pathogens (e.g. Leishmania major and Trypanosoma cruzi). Unusually, these organisms do not regulate their genes by changing the rate at which they are copied into RNA, but by changing the rate of RNA destruction or the rate of translation into protein. We identified an RNA-binding protein, PUF9, responsible for the accumulation of several RNA molecules at a specific time point in the cell division cycle, just after DNA replication. Correspondingly, the proteins encoded by these RNAs appear to function in the division of various cellular structures at this time point or shortly afterwards. Two of them facilitate replication of the kinetoplast (an organelle containing the mitochondrial DNA) while another was found in the mitotic spindle. Their temporal co-expression may stem from another unusual feature of trypanosomes: only one copy of the kinetoplast (and several other organelles) are present per cell, their replication being coordinated with cell division. Indeed, PUF9 may be important in the control of organelle copy-number because suppression of PUF9 resulted in cells with too many kinetoplasts, flagella, or nuclei.
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