Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
Department of Genetics, University of Georgia, Athens, GA 30602, USA.
Nucleic Acids Res. 2024 Jul 8;52(12):6866-6885. doi: 10.1093/nar/gkae392.
The genomes of Leishmania and trypanosomes are organized into polycistronic transcription units flanked by a modified DNA base J involved in promoting RNA polymerase II (Pol II) termination. We recently characterized a Leishmania complex containing a J-binding protein, PP1 protein phosphatase 1, and PP1 regulatory protein (PNUTS) that controls transcription termination potentially via dephosphorylation of Pol II by PP1. While T. brucei contains eight PP1 isoforms, none purified with the PNUTS complex, complicating the analysis of PP1 function in termination. We now demonstrate that the PP1-binding motif of TbPNUTS is required for function in termination in vivo and that TbPP1-1 modulates Pol II termination in T. brucei and dephosphorylation of the large subunit of Pol II. PP1-1 knock-down results in increased cellular levels of phosphorylated RPB1 accompanied by readthrough transcription and aberrant transcription of the chromosome by Pol II, including Pol I transcribed loci that are typically silent, such as telomeric VSG expression sites involved in antigenic variation. These results provide important insights into the mechanism underlying Pol II transcription termination in primitive eukaryotes that rely on polycistronic transcription and maintain allelic exclusion of VSG genes.
利什曼原虫和锥虫的基因组组织成多顺反子转录单元，两侧是参与促进 RNA 聚合酶 II（Pol II）终止的修饰 DNA 碱基 J。我们最近描述了一种包含 J 结合蛋白、PP1 蛋白磷酸酶 1 和 PP1 调节蛋白（PNUTS）的利什曼复合物，该复合物通过 PP1 使 Pol II 去磷酸化，从而潜在地控制转录终止。虽然 T. brucei 含有 8 种 PP1 同工酶，但没有一种与 PNUTS 复合物一起纯化，这使得 PP1 在终止中的功能分析变得复杂。我们现在证明 TbPNUTS 的 PP1 结合基序是体内终止所必需的，并且 TbPP1-1 调节 T. brucei 中的 Pol II 终止和 Pol II 大亚基的去磷酸化。PP1-1 敲低导致磷酸化 RPB1 的细胞水平增加，伴随着通读转录和 Pol II 对染色体的异常转录，包括 Pol I 转录的基因座，这些基因座通常是沉默的，例如参与抗原变异的端粒 VSG 表达位点。这些结果为依赖多顺反子转录并维持 VSG 基因等位基因排斥的原始真核生物中 Pol II 转录终止的机制提供了重要的见解。
