PAP-gamma associates with PAXT nuclear exosome to control the abundance of PROMPT ncRNAs

Pervasive transcription of the human genome generates an abundance of RNAs that must be processed and degraded. The nuclear RNA exosome is the main RNA degradation machinery in the nucleus. However, nuclear exosome must be recruited to its substrates by targeting complexes, such as NEXT or PAXT. By proteomic analysis, we have identified additional subunits of PAXT, including many orthologs of MTREC found in S. pombe. In particular, we show that polyA polymerase gamma was associated with PAXT. Genome-wide mapping of the binding sites of ZFC3H1, RBM26 and PAPgamma, showed that PAXT is recruited to the TSS of hundreds of genes. Loss of ZFC3H1 abolished recruitment of PAXT subunits including PAPgamma to TSSs and concomitantly increased the abundance of PROMPTs at the same sites. Moreover, PAPgamma polyadenylated PROMPTs and modulated their stability. Our results thus provide key insights into the direct targeting and degradation of PROMPT ncRNAs by PAXT, at their genomic sites and depending on polyadenylation of RNAs. Overall design: We used the technology of ChIP-seq to quantify occupancy of PAXT subunits, ZFC3H1, RBM26 and a newly identified subunit, PAPgamma, as well as RNA polymerase II in control cells. Localization of PAXT subunits was also identified by ChIP-seq in cells expressing shRNAs targeting ZFC3F1 or a non-targeting control. We used the RNA-seq technology to quantify RNA expression changes upon loss of PAP-Gamma using siRNA.

人类基因组的广泛转录可产生大量需要经历加工与降解过程的RNA。细胞核RNA外切酶体（nuclear RNA exosome）是细胞核内主要的RNA降解系统。然而，细胞核RNA外切酶体需要通过靶向复合物招募至其底物，例如NEXT或PAXT。通过蛋白质组学分析，我们鉴定出PAXT的新增亚基，包括粟酒裂殖酵母（S. pombe）中MTREC的多个同源蛋白。尤为重要的是，我们证实多聚A聚合酶γ（polyA polymerase gamma）可与PAXT形成关联。对ZFC3H1、RBM26及PAPγ进行全基因组结合位点图谱分析后发现，PAXT会被招募至数百个基因的转录起始位点（transcription start site, TSS）。ZFC3H1的缺失会阻断PAXT亚基（包括PAPγ）向转录起始位点的招募，同时会在相同位点处显著提升启动子上游RNA（PROMPTs）的丰度。此外，PAPγ可对启动子上游RNA进行多聚腺苷酸化修饰，并调控其稳定性。因此，本研究结果为PAXT在其基因组位点上，通过依赖RNA多聚腺苷酸化的机制直接靶向并降解启动子上游非编码RNA（PROMPT ncRNAs）提供了关键理论依据。
实验设计概述：我们采用染色质免疫沉淀测序（ChIP-seq）技术，对对照细胞中PAXT亚基、ZFC3H1、RBM26以及新鉴定的亚基PAPγ与RNA聚合酶Ⅱ的染色质结合占有率进行定量分析。我们还通过ChIP-seq分析了转染靶向ZFC3F1的短发夹RNA（shRNA）或非靶向对照短发夹RNA的细胞中PAXT亚基的定位情况。我们采用RNA测序（RNA-seq）技术，定量分析了通过小干扰RNA（siRNA）敲低PAPγ后细胞内RNA表达水平的变化。