Microprocessor mediates transcription termination in long noncoding microRNA genes

MicroRNA (miRNA) play a major role in the post-transcriptional regulation of gene expression. In mammals most miRNA derive from the introns of protein coding genes where they exist as hairpin structures in the primary gene transcript, synthesized by RNA polymerase II (Pol II). These are cleaved co-transcriptionally by the Microprocessor complex, comprising DGCR8 and the RNase III endonuclease Drosha, to release the precursor (pre-)miRNA hairpin, so generating both miRNA and spliced messenger RNA1-4. However, a substantial minority of miRNA originate from Pol II-synthesized long non coding (lnc) RNA where transcript processing is largely uncharacterized5. Here, we show that most lnc-pri-miRNA do not use the canonical cleavage and polyadenylation (CPA) transcription termination pathway6, but instead use Microprocessor cleavage both to release pre-miRNA and terminate transcription. We present a detailed characterization of one such lnc-pri-miRNA that generates the highly expressed liver-specific miR-1227. Genome-wide analysis then reveals that Microprocessor-mediated transcription termination is commonly used by lnc-pri-miRNA but not by protein coding miRNA genes. This identifies a fundamental difference between lncRNA and pre-mRNA processing. Remarkably, inactivation of the Microprocessor can lead to extensive transcriptional readthrough of lnc-pri-miRNA, resulting in inhibition of downstream genes by transcriptional interference. Consequently we define a novel RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells. Chromatin associated RNA-seq from sicntrl,siDrosha,siDGCR8 treated Hela cells. Same for sicntrl and siDGCR8 from Huh7 cells. Nuclear polyA + and polyA- RNA-seq from sicntrl and siDGCR8 in HeLa cells. Chromatin associated RNA-seq from siDicer treated Hela cells.

微小RNA（MicroRNA, miRNA）在基因表达的转录后调控中发挥核心作用。在哺乳动物体内，大多数miRNA源自蛋白编码基因的内含子区域，它们以发夹结构存在于由RNA聚合酶II（RNA polymerase II, Pol II）合成的初级基因转录本中。这类转录本会由包含DGCR8与RNase III核酸内切酶Drosha的微处理器复合物（Microprocessor complex）进行共转录剪切，释放出前体微小RNA（precursor, pre-miRNA）发夹结构，同时生成miRNA与剪接信使RNA1-4。然而，仍有相当比例的miRNA源自Pol II合成的长链非编码RNA（long non coding, lncRNA），此类转录本的加工机制目前尚未明确5。本研究证实，绝大多数源自长链非编码RNA的初级miRNA（long non-coding primary miRNA, lnc-pri-miRNA）并不依赖经典的剪切与多聚腺苷酸化（cleavage and polyadenylation, CPA）转录终止通路6，而是通过微处理器的剪切同时完成pre-miRNA的释放与转录终止过程。我们对其中一种可高表达肝脏特异性miR-1227的lnc-pri-miRNA开展了详细的特征分析。全基因组层面的分析进一步揭示，微处理器介导的转录终止是lnc-pri-miRNA普遍采用的调控机制，而蛋白编码的miRNA基因则不使用该通路。这一发现明确了长链非编码RNA与前体mRNA（pre-mRNA）加工过程之间的根本性差异。值得注意的是，微处理器的失活会导致lnc-pri-miRNA出现广泛的转录通读现象，进而通过转录干扰抑制下游基因的表达。据此，我们定义了一种全新的、由RNase III介导且不依赖多聚腺苷酸化的哺乳动物细胞Pol II转录终止机制。本研究的配套测序数据集包括：经阴性对照siRNA（sicntrl）、靶向Drosha的siRNA（siDrosha）及靶向DGCR8的siRNA（siDGCR8）处理的HeLa细胞的染色质相关RNA测序数据；经sicntrl与siDGCR8处理的Huh7细胞的对应测序数据；HeLa细胞中经sicntrl与siDGCR8处理的细胞核polyA+与polyA- RNA测序数据；以及经靶向Dicer的siRNA处理的HeLa细胞的染色质相关RNA测序数据。