Ribosomal RNA synthesis by RNA polymerase I is regulated by premature termination of transcription.

The synthesis of the large rRNA precursors is achieved by the RNA polymerase I (Pol I) enzyme. During elongation, Pol I exhibits a highly discontinuous process with a high rate of pauses. Here, we propose that Pol I Premature Termination of Transcription (PTT) is a major regulatory step limiting rRNA production in vivo. We previously isolated a mutant of Pol I carrying the RPA135-F301S mutation. This mutant (hereafter called SuperPol) is able to produce 1.5-fold more rRNA than wild-type (WT). Combining crosslinking and analysis of cDNAs (CRAC) and rRNA analysis we could show that increased rRNA production in SuperPol is associated with a reduced premature release of nascent transcripts and a shift of the polymerase distribution toward the 3’ end of rDNA genes. In vitro, SuperPol shows a defect in nascent transcript cleavage combined with an increased ability to resume elongation after a pause, to the detriment of transcriptional fidelity. SuperPol is resistant to high concentrations of BMH-21, a drug that severely impairs Pol I elongation and triggers the proteasome-mediated degradation of the largest subunits of Pol I, in both human and yeast. Compared to WT, SuperPol retains subunits stability and maintains high levels of Pol I transcription upon BMH-21 treatment. These results show that PTT is both alleviated in SuperPol and targeted by BMH-21 in WT Pol I.大型核糖体RNA（ribosomal RNA, rRNA）前体的合成由RNA聚合酶I（RNA polymerase I, Pol I）催化完成。在转录延伸阶段，Pol I的催化过程呈现高度不连续性，且伴随较高的暂停频率。本文提出，Pol I转录提前终止（Premature Termination of Transcription, PTT）是体内限制rRNA生成的核心调控步骤。我们此前已分离得到携带RPA135-F301S突变的Pol I突变体，该突变体（下文简称SuperPol）的rRNA生成量较野生型（wild-type, WT）提升1.5倍。通过结合交联与cDNA分析（crosslinking and analysis of cDNAs, CRAC）与rRNA分析实验，我们证实SuperPol的rRNA生成量提升与新生转录本的提前释放减少相关，且聚合酶在核糖体DNA（ribosomal DNA, rDNA）基因上的分布向3'端发生偏移。体外实验结果显示，SuperPol存在新生转录本切割缺陷，同时在转录暂停后恢复延伸的能力增强，这一变化以牺牲转录保真度为代价。此外，SuperPol对高浓度BMH-21具有抗性：该药物可严重损害野生型Pol I的延伸过程，并在人类与酵母细胞中诱发蛋白酶体介导的Pol I最大亚基降解。与野生型相比，SuperPol可维持Pol I亚基的稳定性，并在BMH-21处理后仍保持高水平的Pol I转录活性。上述实验结果表明，PTT在SuperPol中得到有效缓解，且BMH-21可靶向作用于野生型Pol I的PTT调控过程。