The trypanosome transcriptome is remodelled during differentiation but displays limited responsiveness within life stages.
The trypanosome transcriptome is remodelled during differentiation but displays limited responsiveness within life stages.
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DOI：
10.1186/1471-2164-9-298
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发表时间：
2008-06-23
期刊：
影响因子：
4.4
通讯作者：
Field MC
中科院分区：
文献类型：
--
作者：
Koumandou VL;Natesan SK;Sergeenko T;Field MC
锥虫利用多顺反子转录来产生绝大多数蛋白质编码 mRNA，这些 mRNA 在缺乏基因特异性启动子的情况下运行。通过聚腺苷酸化和反式剪接对新生转录本的解析，以及特定的 mRNA 周转率，有助于产生稳态转录本水平，其丰度可以在几个数量级上有所不同，并且可以在发育过程中进行调节。我们使用靶向寡核苷酸微阵列，代表强烈发育调节的布氏锥虫膜运输系统和约 10% 的布氏锥虫基因组，来研究阶段间或分化依赖性转录组变化和阶段内灵活性，以应对各种挑战。 6% 的基因群受到发育调控，包括几种小型 GTPases、SNARE、囊泡外壳因子和蛋白激酶，均与之前的数据一致并扩展了之前的数据。因此，锥虫转录组的实质性分化依赖性重塑与膜运输相关。然后使用微阵列和 qRT-PCR 来分析由于特定基因过度表达、敲低、培养条件改变和化学应激而导致的转录组变化。首先，操纵 Rab5 表达会导致网格蛋白表达水平和内吞活性发生协调变化，但稳态 mRNA 水平没有可检测到的变化，这表明该效应是转录后介导的。其次，网格蛋白或变异表面糖蛋白的敲低未能扰乱转录。第三，暴露于二硫苏糖醇或衣霉素没有显示出经典的未折叠蛋白反应的证据，该反应在高等真核生物中通过转录变化介导。最后，血清水平的改变除了转铁蛋白受体 ESAG6/7 表达的变化外，几乎没有引起转录组的改变。虽然锥虫调节 mRNA 丰度以影响伴随分化的主要变化，但给定的分化状态在转录上似乎不灵活。讨论了锥虫中缺乏转录组反应对毒力和生命周期进展模型的影响。
Trypanosomatids utilise polycistronic transcription for production of the vast majority of protein-coding mRNAs, which operates in the absence of gene-specific promoters. Resolution of nascent transcripts by polyadenylation and trans-splicing, together with specific rates of mRNA turnover, serve to generate steady state transcript levels that can differ in abundance across several orders of magnitude and can be developmentally regulated. We used a targeted oligonucleotide microarray, representing the strongly developmentally-regulated T. brucei membrane trafficking system and ~10% of the Trypanosoma brucei genome, to investigate both between-stage, or differentiation-dependent, transcriptome changes and within-stage flexibility in response to various challenges. 6% of the gene cohort are developmentally regulated, including several small GTPases, SNAREs, vesicle coat factors and protein kinases both consistent with and extending previous data. Therefore substantial differentiation-dependent remodeling of the trypanosome transcriptome is associated with membrane transport. Both the microarray and qRT-PCR were then used to analyse transcriptome changes resulting from specific gene over-expression, knockdown, altered culture conditions and chemical stress. Firstly, manipulation of Rab5 expression results in co-ordinate changes to clathrin protein expression levels and endocytotic activity, but no detectable changes to steady-state mRNA levels, which indicates that the effect is mediated post-transcriptionally. Secondly, knockdown of clathrin or the variant surface glycoprotein failed to perturb transcription. Thirdly, exposure to dithiothreitol or tunicamycin revealed no evidence for a classical unfolded protein response, mediated in higher eukaryotes by transcriptional changes. Finally, altered serum levels invoked little transcriptome alteration beyond changes to expression of ESAG6/7, the transferrin receptor. While trypanosomes regulate mRNA abundance to effect the major changes accompanying differentiation, a given differentiated state appears transcriptionally inflexible. The implications of the absence of a transcriptome response in trypanosomes for both virulence and models of life cycle progression are discussed.
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通讯作者：
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