Parast Saeid, Wang Simai, Iwanaszko Marta, He Yue, Olgun Deniz G, Gold Sarah R, Aoi Yuki, Zeidner Jacob M, Howard Benjamin C, Thakur William R, Ramani Vijay, Shilatifard Ali
bioRxiv. 2025 Jun 23:2025.06.23.660200. doi: 10.1101/2025.06.23.660200.
Transcription elongation factors control post-initiation steps of gene expression by RNA polymerase II (RNAPII). We have established distinct mechanistic roles for the essential elongation factors PAF1, NELF, SPT5, SPT6, and the Super Elongaiton Complex (SEC) via acute depletion of each individually in auxin-inducible degron lines. Here, we leverage these degron lines to explore the regulatory intersection of transcription elongation control and pre-mRNA processing. Integrating long- and short-read RNA-seq data to quantify transcript isoform usage at single-molecule resolution, we identify elongation factor-specific RNA processing regulons including a cellular senescence-enriched regulon shared by NELF and SPT6. We then show that long-term depletion of NELF or SPT6 results in reversible growth arrest following early upregulation of a small group of genes, which include the senescence-associated genes CDKN1A (p21) and CCN2. We perform genetic suppressor screens that implicate the elongation factor Elongin A (ELOA) in NELF or SPT6 depletion-induced growth arrest. ELOA loss suppresses NELF depletion-induced pre-mRNA processing defects and the 3' extension of RNAPII occupancy past transcription end sites (TES) at genes induced by NELF depletion. ELOA also occupies TES-proximal regions under normal conditions, and acute ELOA depletion results in a loss of RNAPII processivity at the 3' end of genes, opposing the effects of NELF or SPT6 depletion. Finally, we demonstrate that genetic loss of ELOA confers a growth advantage to aging human primary dermal fibroblasts. These findings establish the existence of novel ELOA-dependent mechanisms regulating transcription maturation, and links these mechanisms to the complex phenomena of cellular senescence and aging.
转录延伸因子通过RNA聚合酶II（RNAPII）控制基因表达的起始后步骤。我们通过在生长素诱导的降解子系中分别急性耗尽每个必需的延伸因子，确定了必需延伸因子PAF1、NELF、SPT5、SPT6和超级延伸复合体（SEC）的不同机制作用。在这里，我们利用这些降解子系来探索转录延伸控制和前体mRNA加工的调控交叉点。整合长读长和短读长RNA测序数据以在单分子分辨率下量化转录本异构体的使用情况，我们鉴定了延伸因子特异性的RNA加工调控子，包括NELF和SPT6共享的富含细胞衰老的调控子。然后我们表明，长期耗尽NELF或SPT6会导致一小群基因早期上调后出现可逆的生长停滞，其中包括衰老相关基因CDKN1A（p21）和CCN2。我们进行了遗传抑制子筛选，发现延伸因子Elongin A（ELOA）与NELF或SPT6耗尽诱导的生长停滞有关。ELOA缺失抑制了NELF耗尽诱导的前体mRNA加工缺陷以及NELF耗尽诱导基因处RNAPII占据在转录终止位点（TES）之后的3'延伸。在正常条件下，ELOA也占据TES近端区域，急性耗尽ELOA会导致基因3'端RNAPII的持续合成能力丧失，这与NELF或SPT6耗尽的影响相反。最后，我们证明ELOA的基因缺失赋予衰老的人类原代表皮成纤维细胞生长优势。这些发现确立了调节转录成熟的新型ELOA依赖性机制的存在，并将这些机制与细胞衰老和老化的复杂现象联系起来。
