Bhandare Pranjali, Narain Ashwin, Hofstetter Julia, Rummel Teresa, Wenzel Julia, Schülein-Völk Christina, Lamer Stephanie, Eilers Ursula, Schlosser Andreas, Eilers Martin, Erhard Florian, Wolf Elmar
Institute of Biochemistry, University of Kiel, Rudolf-Höber-Straße 1, Kiel 24118, Germany.
Cancer Systems Biology Group, Theodor Boveri Institute, University of Würzburg, Am Hubland, Würzburg 97074, Germany.
Nucleic Acids Res. 2025 Feb 8;53(4). doi: 10.1093/nar/gkae1219.
Transcripts produced by RNA polymerase II (RNAPII) are fundamental for cellular responses to environmental changes. It is therefore no surprise that there exist multiple avenues for the regulation of this process. To explore the regulation mediated by RNAPII-interacting proteins, we used a small interfering RNA (siRNA)-based screen to systematically evaluate their influence on RNA synthesis. We identified several proteins that strongly affected RNAPII activity. We evaluated one of the top hits, SCAF1 (SR-related C-terminal domain-associated factor 1), using an auxin-inducible degradation system and sequencing approaches. In agreement with our screen results, acute depletion of SCAF1 decreased RNA synthesis, and showed an increase of Serine-2 phosphorylated-RNAPII (pS2-RNAPII). We found that the accumulation of pS2-RNAPII within the gene body occurred at GC-rich regions and was indicative of stalled RNAPII complexes. The accumulation of stalled RNAPII complexes was accompanied by reduced recruitment of initiating RNAPII, explaining the observed global decrease in transcriptional output. Furthermore, upon SCAF1 depletion, RNAPII complexes showed increased association with components of the proteasomal-degradation machinery. We concluded that in cells lacking SCAF1, RNAPII undergoes a rather interrupted passage, resulting in intervention by the proteasomal-degradation machinery to clear stalled RNAPII. While cells survive the compromised transcription caused by absence of SCAF1, further inhibition of proteasomal-degradation machinery is synthetically lethal.
RNA聚合酶II（RNAPII）产生的转录本对于细胞对环境变化的反应至关重要。因此，存在多种调控这一过程的途径也就不足为奇了。为了探索由与RNAPII相互作用的蛋白质介导的调控，我们使用了基于小干扰RNA（siRNA）的筛选方法来系统地评估它们对RNA合成的影响。我们鉴定出了几种强烈影响RNAPII活性的蛋白质。我们使用生长素诱导降解系统和测序方法评估了排名靠前的命中蛋白之一SCAF1（SR相关C末端结构域相关因子1）。与我们的筛选结果一致，SCAF1的急性耗竭降低了RNA合成，并显示丝氨酸2磷酸化的RNAPII（pS2-RNAPII）增加。我们发现基因体内pS2-RNAPII的积累发生在富含GC的区域，这表明RNAPII复合物停滞。停滞的RNAPII复合物的积累伴随着起始RNAPII募集的减少，这解释了观察到的转录输出的整体下降。此外，在SCAF1耗竭后，RNAPII复合物与蛋白酶体降解机制的成分的结合增加。我们得出结论，在缺乏SCAF1的细胞中，RNAPII经历了相当中断的过程，导致蛋白酶体降解机制进行干预以清除停滞的RNAPII。虽然细胞在因缺乏SCAF1而受损的转录中存活下来，但对蛋白酶体降解机制的进一步抑制具有合成致死性。
