Determining the effect of RPA49 on RNA polymerase I occupancy in vivo

The central goal of this project was to determine the role of the RNA polymerase I (Pol I) subunits RPA34 and RPA49 in rRNA synthesis. Previous studies by various groups have demonstrated that these two subunits may play a role in transcription initiation and elongation by on Pol I occupancy in vivo, but this is still not clearly defined. Here, we deployed a high-resolution technique, native elongating transcript sequencing (NET-seq), to test the effects of RPA34 and RPA49 on Pol I occupancy in vivo. We found that when RPA34 was deleted, there was a significant reduction in Pol I occupancy across the rDNA template. Additionally, when RPA49 was deleted, Pol I occupancy was even further reduced across the template. Collectively, our findings suggest that perhaps RPA34 acts to stabilize RPA49 and these subunits may act primarily as transcription initiation factors for Pol I.本研究的核心目标是明确RNA聚合酶I（RNA polymerase I, Pol I）的亚基RPA34与RPA49在核糖体RNA（rRNA）合成中的作用。此前多个研究团队已证实，这两个亚基可能通过体内RNA聚合酶I的模板占据参与转录起始与延伸过程，但具体机制仍未明确。本研究采用高分辨率技术——新生延伸转录本测序（native elongating transcript sequencing, NET-seq），探究RPA34与RPA49对体内RNA聚合酶I模板占据情况的影响。实验结果显示，敲除RPA34后，核糖体DNA（rDNA）模板上的RNA聚合酶I占据量显著降低；此外，敲除RPA49后，模板上的RNA聚合酶I占据量进一步下降。综合以上发现，本研究表明RPA34可能起到稳定RPA49的作用，且这两个亚基主要作为RNA聚合酶I的转录起始因子发挥功能。