In most organisms, the maturation of nascent RNAs is coupled to transcription, undergoing many processing steps co-transcriptionally. Unlike in animals, the RNA polymerase II (RNAPII) transcribes microRNAs (miRNAs) as long and structurally variable pri-miRNAs in plants. Current evidence suggests that the miRNA biogenesis complex assembly initiates early during the transcription of pri-miRNAs in plants. However, it is unknown whether miRNA processing occurs co-transcriptionally. Here, we show that plant miRNA biogenesis is coupled to transcription in a process that relies on the formation of DNA:RNA hybrids (R-loops) between the nascent transcript and the encoding loci. We used native elongating transcript sequencing data and imaging techniques to demonstrate that plant miRNA biogenesis occurs co-transcriptionally. We found that the entire biogenesis occurs coupled to transcription for pri-miRNAs processed from the loop but requires a second nucleoplasmic step for those processed from the base of the hairpin. Furthermore, we found that co- and post-transcriptional miRNA processing mechanisms co-exist for most miRNAs in a dynamic balance. Notably, we discovered that R-loops between the 5’-end single-stranded arm of the pri-miRNAs and the encoding loci anchor the transcript, promoting co-transcriptional processing. Our data demonstrate the coupling of transcription and miRNA processing in plants and discovered an unexpected function for R-loops promoting RNA processing. Furthermore, our results suggest the neo-functionalization of co-transcriptionally processed miRNAs, boosting countless regulatory scenarios.

中文翻译：

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新生的 pri-miRNAs 和编码基因座之间的 R-Loops 促进植物中 miRNAs 的共转录加工

在大多数生物体中，新生 RNA 的成熟与转录相关，经过许多共同转录的加工步骤。与动物不同，RNA 聚合酶 II (RNAPII) 将微小 RNA (miRNA) 转录为植物中长且结构可变的 pri-miRNA。目前的证据表明 miRNA 生物发生复合体组装在植物中 pri-miRNA 的转录过程中开始。然而，尚不清楚 miRNA 加工是否与共转录一起发生。在这里，我们展示了植物 miRNA 生物发生在依赖于新生转录物和编码基因座之间形成 DNA:RNA 杂交（R 环）的过程中与转录相结合。我们使用天然延伸转录本测序数据和成像技术来证明植物 miRNA 生物发生是共转录的。我们发现整个生物发生与从环加工的 pri-miRNA 的转录耦合，但对于从发夹底部加工的那些，需要第二个核质步骤。此外，我们发现大多数 miRNA 的共转录和转录后 miRNA 加工机制以动态平衡共存。值得注意的是，我们发现 pri-miRNA 的 5' 端单链臂和编码基因座之间的 R 环锚定转录物，促进共转录加工。我们的数据证明了植物中转录和 miRNA 加工的耦合，并发现了 R-loops 促进 RNA 加工的意外功能。此外，我们的结果表明共转录处理的 miRNA 的新功能化，促进了无数的监管场景。