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Rapid block of pre-mRNA splicing by chemical inhibition of analog-sensitive CRK9 in Trypanosoma brucei.
Molecular Microbiology ( IF 2.6 ) Pub Date : 2020-02-18 , DOI: 10.1111/mmi.14489 Ujwala Gosavi 1 , Ankita Srivastava 1 , Nitika Badjatia 1 , Arthur Günzl 1
Molecular Microbiology ( IF 2.6 ) Pub Date : 2020-02-18 , DOI: 10.1111/mmi.14489 Ujwala Gosavi 1 , Ankita Srivastava 1 , Nitika Badjatia 1 , Arthur Günzl 1
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Trypanosoma brucei CRK9 is an essential cyclin‐dependent kinase for the parasite‐specific mode of pre‐mRNA processing. In trypanosomes, protein coding genes are arranged in directional arrays that are transcribed polycistronically, and individual mRNAs are generated by spliced leader trans ‐splicing and polyadenylation, processes that are functionally linked. Since CRK9 silencing caused a decline of mRNAs, a concomitant increase of unspliced pre‐mRNAs and the disappearance of the trans ‐splicing Y structure intermediate, CRK9 is essential for the first step of splicing. CRK9 depletion also caused a loss of phosphorylation in RPB1, the largest subunit of RNA polymerase (pol) II. Here, we established cell lines that exclusively express analog‐sensitive CRK9 (CRK9AS). Inhibition of CRK9AS in these cells by the ATP‐competitive inhibitor 1‐NM‐PP1 reproduced the splicing defects and proved that it is the CKR9 kinase activity that is required for pre‐mRNA processing. Since defective trans‐ splicing was detected as early as 5 min after inhibitor addition, CRK9 presumably carries out reversible phosphorylation on the pre‐mRNA processing machinery. Loss of RPB1 phosphorylation, however, took 12–24 hr. Surprisingly, RNA pol II‐mediated RNA synthesis in 24 hr‐treated cells was upregulated, indicating that, in contrast to other eukaryotes, RPB1 phosphorylation is not a prerequisite for transcription in trypanosomes.
中文翻译：
通过化学抑制布氏锥虫中类似物敏感的 CRK9 快速阻断前 mRNA 剪接。
布氏锥虫CRK9 是一种重要的细胞周期蛋白依赖性激酶，用于寄生虫特异性前 mRNA 加工模式。在锥虫中，蛋白质编码基因排列成多顺反子转录的定向阵列，并且通过剪接的前导反式剪接和聚腺苷酸化（功能上相连的过程）生成单个 mRNA。由于CRK9沉默导致 mRNA 减少，随之而来的是未剪接前 mRNA 的增加以及反式剪接 Y 结构中间体的消失，因此 CRK9 对于剪接的第一步至关重要。 CRK9 耗竭还导致 RPB1（RNA 聚合酶 (pol) II 的最大亚基）磷酸化丧失。在这里，我们建立了专门表达模拟敏感 CRK9 (CRK9 AS ) 的细胞系。 ATP 竞争性抑制剂 1-NM-PP1 对这些细胞中的 CRK9 AS的抑制再现了剪接缺陷，并证明前 mRNA 加工所需的是 CKR9 激酶活性。由于早在添加抑制剂后 5 分钟就检测到有缺陷的转拼，CRK9 可能在前 mRNA 加工机器上进行可逆磷酸化。然而，RPB1 磷酸化的丧失需要 12-24 小时。令人惊讶的是，24 小时处理的细胞中 RNA pol II 介导的 RNA 合成上调，这表明与其他真核生物相比，RPB1 磷酸化并不是锥虫中转录的先决条件。
更新日期：2020-02-18
中文翻译：
通过化学抑制布氏锥虫中类似物敏感的 CRK9 快速阻断前 mRNA 剪接。
布氏锥虫CRK9 是一种重要的细胞周期蛋白依赖性激酶，用于寄生虫特异性前 mRNA 加工模式。在锥虫中，蛋白质编码基因排列成多顺反子转录的定向阵列，并且通过剪接的前导反式剪接和聚腺苷酸化（功能上相连的过程）生成单个 mRNA。由于CRK9沉默导致 mRNA 减少，随之而来的是未剪接前 mRNA 的增加以及反式剪接 Y 结构中间体的消失，因此 CRK9 对于剪接的第一步至关重要。 CRK9 耗竭还导致 RPB1（RNA 聚合酶 (pol) II 的最大亚基）磷酸化丧失。在这里，我们建立了专门表达模拟敏感 CRK9 (CRK9 AS ) 的细胞系。 ATP 竞争性抑制剂 1-NM-PP1 对这些细胞中的 CRK9 AS的抑制再现了剪接缺陷，并证明前 mRNA 加工所需的是 CKR9 激酶活性。由于早在添加抑制剂后 5 分钟就检测到有缺陷的转拼，CRK9 可能在前 mRNA 加工机器上进行可逆磷酸化。然而，RPB1 磷酸化的丧失需要 12-24 小时。令人惊讶的是，24 小时处理的细胞中 RNA pol II 介导的 RNA 合成上调，这表明与其他真核生物相比，RPB1 磷酸化并不是锥虫中转录的先决条件。
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