Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble
Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble
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DOI：
10.1038/s41467-019-10780-2
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发表时间：
2019-07-05
影响因子：
16.6
通讯作者：
Doudna, Jennifer A.
中科院分区：
文献类型：
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作者：
Liu, Tina Y.;Liu, Jun-Jie;Doudna, Jennifer A.
III-A型CRISPR-Cas系统是原核RNA引导的适应性免疫系统，其使用蛋白质-RNA复合物Csm用于针对外源DNA的转录依赖性免疫。CSM可以切割RNA和单链DNA（ssDNA），但它是否靶向转录延伸过程中的一个或两个核酸是未知的。在这里，我们表明，结合的Thermos thermophilus（T。在转录延伸复合物（TEC）中，嗜热菌（thermophilus）Csm（TthCsm）与新生转录物的结合促进TthCsm与RNA聚合酶（RNAP）的束缚而不是直接接触。生化实验表明，TthCsm和表皮葡萄球菌（S。epidermidis）Csm（SepCsm）在转录泡处切割RNA转录物，但不切割ssDNA。综上所述，这些结果表明，III型系统主要靶向转录本，而不是TEC中解旋的ssDNA，用于针对双链DNA（dsDNA）引物和质粒的免疫。这揭示了Csm和真核RNA干扰之间的相似性，真核RNA干扰也使用RNA引导的RNA靶向来沉默活跃转录的基因。
Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermos thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.