Chromatin-Associated Protein Complexes Link DNA Base J and Transcription Termination in Leishmania.
Chromatin-Associated Protein Complexes Link DNA Base J and Transcription Termination in Leishmania.
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DOI：
10.1128/msphere.01204-20
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发表时间：
2021-02-24
期刊：
影响因子：
4.8
通讯作者：
Myler PJ
中科院分区：
文献类型：
--
作者：
Jensen BC;Phan IQ;McDonald JR;Sur A;Gillespie MA;Ranish JA;Parsons M;Myler PJ
与大多数其他真核生物不同，利什曼原虫和其他锥虫原生动物在很大程度上避开了基因表达的转录控制，而是依赖于来自多顺反子转录单位（PTU）的mRNA的转录后调控。在这些寄生虫中，称为J的新型修饰核苷酸碱基（β-d-吡喃葡萄糖基氧基甲基尿嘧啶）在确保转录终止仅发生在每个PTU的末端而不是单个基因的多聚腺苷酸化位点方面起着关键作用。为了进一步了解J相关过程的生物学，我们使用串联亲和纯化（TAP）标记和质谱来揭示与葡萄糖基转移酶相互作用的蛋白质，该葡萄糖基转移酶在J合成中执行最后一步。这些研究确定了四种蛋白质，它们使人联想到PTW/PP 1复合物中的亚基，该复合物控制高等真核生物中的转录终止。此外，生物信息学分析确定了利什曼原虫PTW/PP 1的DNA结合亚基作为一种新的J结合蛋白（JBP 3），这也是另一种含有蛋白质的复合物的一部分，该蛋白质具有暗示在染色质修饰/重塑中起作用的结构域。此外，JBP 3与PAF 1复合物的锥虫等同物相关联（尽管是瞬时和/或间接的），PAF 1复合物参与其他真核生物中转录的调节。利什曼原虫JBP 3表达水平的下调导致大多数PTU 3′端转录通读的显著增加。我们认为JBP 3将这些复合物中的一个或多个募集到PTU末端的含J区域，在那里它们阻止RNA聚合酶的进展。这种转录终止与单个基因剪接的解耦使得寄生虫能够独特地依赖于基因表达的多顺反子转录和转录后调节。利什曼原虫引起多种严重的人类疾病，没有有效的疫苗，并对目前的药物治疗产生耐药性。我们以前已经表明，一种新的DNA碱基称为J是关键的多顺反子基因簇，是利什曼原虫和相关锥虫的标志的末端转录终止。在这里，我们描述了一种新的J-结合蛋白（JBP 3）与三种不同的蛋白质复合物，这让人想起那些参与在其他真核生物中的转录控制。然而，寄生虫复合物已被重新编程，以调节锥虫中的转录和基因表达与哺乳动物宿主不同，为开发针对这些重要病原体的新型化疗药物提供了新的机会。
Unlike most other eukaryotes, Leishmania and other trypanosomatid protozoa have largely eschewed transcriptional control of gene expression, relying instead on posttranscriptional regulation of mRNAs derived from polycistronic transcription units (PTUs). In these parasites, a novel modified nucleotide base (β-d-glucopyranosyloxymethyluracil) known as J plays a critical role in ensuring that transcription termination occurs only at the end of each PTU, rather than at the polyadenylation sites of individual genes. To further understand the biology of J-associated processes, we used tandem affinity purification (TAP) tagging and mass spectrometry to reveal proteins that interact with the glucosyltransferase performing the final step in J synthesis. These studies identified four proteins reminiscent of subunits in the PTW/PP1 complex that controls transcription termination in higher eukaryotes. Moreover, bioinformatic analyses identified the DNA-binding subunit of Leishmania PTW/PP1 as a novel J-binding protein (JBP3), which is also part of another complex containing proteins with domains suggestive of a role in chromatin modification/remodeling. Additionally, JBP3 associates (albeit transiently and/or indirectly) with the trypanosomatid equivalent of the PAF1 complex involved in the regulation of transcription in other eukaryotes. The downregulation of JBP3 expression levels in Leishmania resulted in a substantial increase in transcriptional readthrough at the 3′ end of most PTUs. We propose that JBP3 recruits one or more of these complexes to the J-containing regions at the end of PTUs, where they halt the progression of the RNA polymerase. This decoupling of transcription termination from the splicing of individual genes enables the parasites’ unique reliance on polycistronic transcription and posttranscriptional regulation of gene expression. IMPORTANCE Leishmania parasites cause a variety of serious human diseases, with no effective vaccine and emerging resistance to current drug therapy. We have previously shown that a novel DNA base called J is critical for transcription termination at the ends of the polycistronic gene clusters that are a hallmark of Leishmania and related trypanosomatids. Here, we describe a new J-binding protein (JBP3) associated with three different protein complexes that are reminiscent of those involved in the control of transcription in other eukaryotes. However, the parasite complexes have been reprogrammed to regulate transcription and gene expression in trypanosomatids differently than in the mammalian hosts, providing new opportunities to develop novel chemotherapeutic treatments against these important pathogens.