Gene expression and alternative splicing profile of MFAP1- and THOC1-depleted human cells

THO/TREX is a conserved complex with a role in messenger ribonucleoprotein biogenesis that links gene expression and genome instability. Here we show that human THO interacts with MFAP1, a spliceosome-associated factor. Interestingly, MFAP1 depletion impairs cell proliferation and genome integrity, increasing γH2AX foci and DNA breaks. This phenotype is not dependent either on transcription or RNA-DNA hybrids. Mutations in the yeast orthologous gene SPP381, also confer a similar transcription-independent genome instability supporting a conserved role. MFAP1 depletion has a wide effect on splicing and gene expression in human cells, determined by transcriptome analyses, that affects a number of DNA damage response (DDR) genes, which supports an indirect role of MFAP1 on genome integrity. Our work defines a novel functional interaction between THO and RNA processing and argues that splicing factors may contribute to genome integrity indirectly by regulating the expression of DDR genes rather than by a direct role. HeLa cells were depleted of MFAP1 or THOC1 by siRNA during 72 h and their global gene expression and alternative splicing profiles were analyzed. Data were used to describe genes that were up- or down-regulated or showed alternative splicing changes after MFAP1 or THOC1 depletion. We analyzed RNA from control cells (siC), MFAP1-depleted cells (siMFAP1) and THOC1-depleted cells (siTHOC1). For each condition, microarray analysis was conducted in triplicate from three different biological replicates (Total = 9 samples). We compared siMFAP1 vs siC, siTHOC1 vs siC and siMFAP1 vs siTHOC1.

THO/TREX是一类保守的复合物，参与信使核糖核蛋白（messenger ribonucleoprotein）生物发生过程，可介导基因表达与基因组不稳定之间的关联。本研究证实，人类THO复合物与剪接体相关因子MFAP1存在相互作用。值得注意的是，MFAP1敲低会损害细胞增殖与基因组完整性，增加γH2AX焦点（γH2AX foci）数量与DNA断裂数目。该表型既不依赖于转录过程，也不依赖于RNA-DNA杂交体（RNA-DNA hybrids）。酵母同源基因SPP381的突变同样可引发类似的转录非依赖性基因组不稳定，印证了该功能的保守性。转录组分析显示，人类细胞中MFAP1敲低对剪接与基因表达具有广泛影响，波及大量DNA损伤应答（DNA damage response, DDR）基因，这为MFAP1通过间接途径影响基因组完整性提供了实验依据。本研究阐明了THO复合物与RNA加工过程间的新型功能互作，并提出：剪接因子可通过调控DNA损伤应答基因的表达，而非直接作用于基因组，间接参与维持基因组完整性。

研究人员通过小干扰RNA（siRNA）在海拉（HeLa）细胞中分别敲低MFAP1与THOC1，持续处理72小时后，分析其全局基因表达与可变剪接谱。实验数据用于描述MFAP1或THOC1敲低后出现上调、下调或可变剪接改变的基因。本研究收集了对照组细胞（siC）、MFAP1敲低细胞（siMFAP1）与THOC1敲低细胞（siTHOC1）的RNA。针对每种实验条件，均通过3次生物学重复完成微阵列分析，总计9个样本。我们对以下三组对照进行了比较：siMFAP1 vs siC、siTHOC1 vs siC以及siMFAP1 vs siTHOC1。