Unlike most other eukaryotes, Leishmania and other trypanosomatid protozoa have largely eschewed transcriptional control of gene expression; relying instead on post-transcriptional 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 regulation of transcription in other eukaryotes. Down-regulation of JBP3 expression levels in Leishmania resulted in a substantial increase in transcriptional read-through 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 progression of the RNA polymerase. This de-coupling of transcription termination from splicing of individual genes enables the parasites' unique reliance on polycistronic transcription and post-transcriptional regulation of gene expression.

中文翻译：

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染色质相关蛋白复合物连接利什曼原虫的DNA碱基J和转录终止

与大多数其他真核生物不同，利什曼原虫和其他锥虫原虫在很大程度上避开了基因表达的转录控制。而是依赖转录后调控的多顺反子转录单位（PTU）衍生的mRNA。在这些寄生虫中，被称为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在每个PTU的末端而不是单个基因的聚腺苷酸化位点发挥关键作用。为了进一步了解J相关过程的生物学特性，我们使用了串联亲和纯化（TAP-tagging）和质谱技术来揭示与糖基转移酶相互作用的蛋白，从而完成J合成的最后一步。这些研究发现了四种蛋白质，它们让人联想到PTW / PP1复合物中的亚基，该复合物可控制高级真核生物中的转录终止。此外，生物信息学分析鉴定了DNA的DNA结合亚基利什曼原虫PTW / PP1是一种新型的J结合蛋白（JBP3），它也是另一种复合物的一部分，该复合物包含具有暗示在染色质修饰/重塑中起作用的结构域的蛋白质。另外，JBP3（尽管是暂时和/或间接地）与参与其他真核生物转录调控的PAF1复合物的锥虫等同。利什曼原虫中JBP3表达水平的下调导致大多数PTU的3'端转录阅读率大幅增加。我们建议JBP3在PTU末端将一种或多种这些复合物募集到含J的区域，在那里它们停止RNA聚合酶的进程。转录终止与单个基因剪接的解偶联使寄生虫的