TAF2 condensation in nuclear speckles links basal transcription factor TFIID to RNA splicing factors [RNA-Seq]

TFIID is an essential basal transcription factor, crucial for RNA polymerase II (pol II) promoter recognition and transcription initiation. The TFIID complex consists of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs) that contain intrinsically disordered regions (IDRs) with currently unknown functions. Here, we show that a conserved IDR drives TAF2 to nuclear speckle condensates independently of other TFIID subunits. Quantitative mass spectrometry analyses reveal TAF2 proximity to RNA splicing factors including specific interactions of the TAF2 IDR with SRRM2 in nuclear speckles. Deleting the IDR from TAF2 does not majorly impact global gene expression but results in changes of alternative splicing events. Further, genome-wide binding analyses suggest that the TAF2 IDR impedes TAF2 promoter association by guiding TAF2 to nuclear speckles. This study demonstrates that an IDR within the large multiprotein complex TFIID controls nuclear compartmentalization and thus links distinct molecular processes, namely transcription initiation and RNA splicing. Overall design: To investigate gene expression changes and alternative splicing events of GFP-NLS-TAF2deltaIDR and GFP-TAF2 expressing cells, we performed RNA sequencing (RNA-seq). HeLa Flp-In T-Rex cells harboring a transgene under the control of a doxycycline-inducible promoter were used to express GFP-NLS-TAF2deltaIDR and GFP-TAF2 with 1 ug/mL doxycycline for 17.5 h. Control cells without transgene were also induced with 1 ug/mL doxycycline. The experiment was performed as technical triplicate.

转录因子IID（TFIID）是一类必需的基础转录因子，在RNA聚合酶II（RNA polymerase II，pol II）的启动子识别与转录起始过程中发挥关键作用。TFIID复合物由TATA盒结合蛋白（TATA binding protein，TBP）与13个TBP相关因子（TBP-associated factors，TAFs）组成，这些TAFs均携带有目前功能尚不明确的内在无序区域（intrinsically disordered regions，IDRs）。本研究证实，一段保守的内在无序区域可独立于其他TFIID亚基，将TAF2招募至核斑凝聚体中。定量蛋白质质谱分析结果显示，TAF2与RNA剪接因子存在邻近关联，其中TAF2的内在无序区域与核斑中的SRRM2存在特异性相互作用。敲除TAF2的内在无序区域不会对全局基因表达产生显著影响，但会导致可变剪接事件发生改变。此外，全基因组结合分析表明，TAF2的内在无序区域通过将TAF2引导至核斑，阻碍了TAF2与启动子的结合。本研究揭示，大型多蛋白复合物TFIID内部的一段内在无序区域可调控细胞核区室化，从而将转录起始与RNA剪接这两个独立的分子过程关联起来。

整体实验设计：为探究表达GFP-NLS-TAF2ΔIDR与GFP-TAF2的细胞的基因表达变化及可变剪接事件，本研究开展了RNA测序（RNA sequencing，RNA-seq）实验。我们使用携带有受多西环素（doxycycline）诱导型启动子调控的转基因的HeLa Flp-In T-Rex细胞系，以1 μg/mL多西环素诱导其表达GFP-NLS-TAF2ΔIDR与GFP-TAF2，诱导时长为17.5小时；同时设置未携带转基因的对照细胞，同样以1 μg/mL多西环素进行诱导。本实验设置3次技术重复。