Precise regulation of transcription by RNA polymerase II (RNAPII) is critical for organismal growth and development. However, what determines whether an engaged RNAPII will synthesize a full-length transcript or terminate prematurely is poorly understood. Notably, RNAPII is far more susceptible to termination when transcribing non-coding RNAs than when synthesizing protein-coding mRNAs, but the mechanisms underlying this are unclear. To investigate the impact of transcribed sequence on elongation potential, we developed a method to screen the effects of thousands of INtegrated Sequences on Expression of RNA and Translation using high-throughput sequencing (INSERT-seq). We found that higher AT content in non-coding RNAs, rather than specific sequence motifs, drives RNAPII termination. Further, we demonstrate that 5′ splice sites autonomously stimulate processive transcription, even in the absence of polyadenylation signals. Our results reveal a potent role for the transcribed sequence in dictating gene output and demonstrate the power of INSERT-seq toward illuminating these contributions.

RNA 聚合酶 II (RNAPII) 对转录的精确调控对于生物体的生长和发育至关重要。然而，人们对决定参与的 RNAPII 是否会合成全长转录本或过早终止的因素知之甚少。值得注意的是，RNAPII 在转录非编码 RNA 时比在合成蛋白质编码 mRNA 时更容易终止，但其背后的机制尚不清楚。为了研究转录序列对延伸潜力的影响，我们开发了一种方法，使用高通量测序 (INSERT-seq) 筛选数千个整合序列对 RNA 表达和翻译的影响。我们发现非编码 RNA 中更高的 AT 含量，而不是特定的序列基序，驱动 RNAPII 终止。更远，我们证明 5' 剪接位点会自动刺激进行性转录，即使在没有聚腺苷酸化信号的情况下也是如此。我们的结果揭示了转录序列在控制基因输出方面的强大作用，并证明了 INSERT-seq 对阐明这些贡献的力量。