Enhancement of Transcription by a Splicing-Competent Intron Is Dependent on Promoter Directionality

Enhancement of transcription by a splicing-competent intron is an evolutionarily conserved feature among eukaryotes. The molecular mechanism underlying the phenomenon, however, is not entirely clear. Here we show that the intron is an important regulator of promoter directionality. Employing strand-specific transcription run-on (TRO) analysis, we show that the transcription of mRNA is favored over the upstream anti-sense transcripts (uaRNA) initiating from the promoter in the presence of an intron. Mutation of either the 5′ or 3′ splice site resulted in the reversal of promoter directionality, thereby suggesting that it is not merely the 5′ splice site but the entire splicing-competent intron that regulates transcription directionality. ChIP analysis revealed the recruitment of termination factors near the promoter region in the presence of an intron. Removal of intron or the mutation of splice sites adversely affected the promoter localization of termination factors. We have earlier demonstrated that the intron-mediated enhancement of transcription is dependent on gene looping. Here we show that gene looping is crucial for the recruitment of termination factors in the promoter-proximal region of an intron-containing gene. In a looping-defective mutant, despite normal splicing, the promoter occupancy of factors required for poly(A)-dependent termination of transcription was compromised. This was accompanied by a concomitant loss of transcription directionality. On the basis of these results, we propose that the intron-dependent gene looping places the terminator-bound factors in the vicinity of the promoter region for termination of the promoter-initiated upstream antisense transcription, thereby conferring promoter directionality.具备剪接能力的内含子对转录的增强作用是真核生物中进化保守的特征之一。然而，该现象背后的分子机制尚未完全阐明。本研究证实，内含子是调控启动子方向性的重要因子。通过链特异性转录运行检测（strand-specific transcription run-on, TRO）分析，我们发现当基因携带内含子时，mRNA的转录相较于由该启动子起始的上游反义转录本（upstream anti-sense transcripts, uaRNA）更具优势。对5'或3'剪接位点进行突变，会导致启动子方向性发生逆转，这表明调控转录方向性的并非仅5'剪接位点，而是完整的具备剪接能力的内含子。染色质免疫共沉淀（ChIP）分析显示，当存在内含子时，转录终止因子会在启动子区域附近被招募。去除内含子或突变剪接位点，会对终止因子在启动子区域的定位产生负面影响。我们此前的研究已证实，内含子介导的转录增强作用依赖于基因成环。本研究进一步发现，基因成环对于在携带内含子的基因的启动子近端区域招募终止因子至关重要。在成环缺陷型突变体中，尽管剪接过程正常，但参与转录多聚腺苷酸依赖型终止的相关因子的启动子结合水平受到损害。这一现象同时伴随着转录方向性的同步丧失。基于上述实验结果，我们提出如下假说：内含子依赖型基因成环将结合于转录终止位点的终止因子募集至启动子区域附近，以终止由启动子起始的上游反义转录，从而赋予启动子方向性。