Prokaryotic silencing (psi) RNAs in Pyrococcus furiosus
Prokaryotic silencing (psi) RNAs in Pyrococcus furiosus
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DOI：
10.1261/rna.1246808
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发表时间：
2008-12-01
期刊：
影响因子：
4.5
通讯作者：
Terns, Michael P.
中科院分区：
文献类型：
--
作者：
Hale, Caryn;Kleppe, Kyle;Terns, Michael P.
在许多原核生物中，现在认为由群集的常规散布的短文重复（CRISPR）基因座引起的非编码RNA被认为可以通过RNAi样途径介导对病毒和其他分子入侵者的防御。 CRISPR基因座包含多个与入侵序列相似的短区域，该序列被短序列分开，与相应病毒对感染的抗性有关。假设源自这些区域的RNA被称为原核生物沉默（PSI）RNA，引导伴侣蛋白的类似切片板的复合物，以破坏入侵者核酸。在这里，我们调查了CRISPR衍生的RNA在Furiosus的古怪pyrococcus中。北部分析显示，多种RNA物种与提出的生物发生途径一致，该途径包括全长CRISPR基因座转录和中间介质，由内核溶解裂解在重复序列中产生。但是，我们的结果将CRISPR基因座的主要产物确定为小psiRNA，主要由靶向靶向序列组成，可能只有5-10个CRISPR重复序列的核苷酸。这些RNA是狂热疟原虫中最丰富的CRISPR RNA物种，很可能是拟议的原核生物RNAi（PRNAI）系统的效应络合物的指南。我们分析了在非换性条件下分离的无细胞提取物，发现各种CRISPR RNA物种是不同RNA-蛋白质复合物的组成部分，其中包括至少两个包含成熟长度PSIRNA的复合物。最后，RNA是由P. furiosus基因组中的所有七个CRISPR基因座产生的，有趣的是，最近获得的psirnas编码了CRISPR基因座的领导者序列近端似乎是最丰富的。
In many prokaryotes, noncoding RNAs that arise from the clustered regularly interspaced short palindromic repeat (CRISPR) loci are now thought to mediate defense against viruses and other molecular invaders by an RNAi-like pathway. CRISPR loci contain multiple short regions of similarity to invader sequences separated by short repeat sequences, and are associated with resistance to infection by corresponding viruses. It is hypothesized that RNAs derived from these regions, termed prokaryotic silencing (psi) RNAs, guide Slicer-like complexes of partner proteins to destroy invader nucleic acids. Here we have investigated CRISPR-derived RNAs in the archaeon Pyrococcus furiosus. Northern analysis revealed multiple RNA species consistent with a proposed biogenesis pathway that includes full-length CRISPR locus transcripts and intermediates generated by endonucleolytic cleavages within the repeat sequences. However, our results identify the principal products of the CRISPR loci as small psiRNAs comprised primarily of invader-targeting sequence with perhaps only 5-10 nucleotides of CRISPR repeat sequence. These RNAs are the most abundant CRISPR RNA species in P. furiosus and are likely the guides for the effector complexes of the proposed prokaryotic RNAi (pRNAi) system. We analyzed cell-free extracts fractionated under non-denaturing conditions and found that the various CRISPR RNA species are components of distinct RNA-protein complexes, including at least two complexes that contain mature-length psiRNAs. Finally, RNAs are produced from all seven CRISPR loci present in the P. furiosus genome, and interestingly, the most recently acquired psiRNAs encoded proximal to the leader sequence of a CRISPR locus appear to be the most abundant.