Identification and Characterization of Small RNAs in the Hyperthermophilic Archaeon <em>Sulfolobus solfataricus</em>

The term RNA silencing (RNA interference, RNAi) describes a set of mechanisms that regulate gene expression in eukaryotes. Small interfering RNAs (siRNA) and microRNAs (miRNAs) are two major types of RNAi-associated small RNAs (smRNAs) found in most eukaryotic organisms. Despite the presence of a plethora of non-coding RNAs longer than 50-nucleotide (nt) in length in various species of Archaea, little is known about smRNAs in archaea that resemble the 20–24-nt long smRNAs found in eukaryotes, which have been implicated in the post-transcriptional control of gene expression. Here, we report the finding of a large number of smRNAs approximatelly 20-nt in length, including phased smRNAs and potential miRNAs, from the hyperthermophilic archaeon Sulfolobus solfataricus p2 (Ssp2) based on deep sequencing. The expression of some of the miRNA candidates in Ssp2 was confirmed. Consistent with the Ssp2 hyperthermophilic properties, we found that higher temperatures more efficiently induced the production of the miRNA candidates in an in vitro system using the putative foldback precursor transcripts incubated with Ssp2 extract. Although we initially predicted putative target genes of some miRNA candidates, further analysis mapped the cleavage sites downstream of the miRNA candidate complementary regions, similar to those involved in plant miRNA-mediated TAS transcript cleavage. We also identified smRNAs from clustered, regularly interspaced, short palindromic repeat (CRISPR) loci, which play important roles in prokaryotic microbial defense systems. Archaea represent a unique life form next to Bacteria and Eukarya, and our results may provide a useful resource for further in-depth study on the regulation and evolution of smRNAs in this special organism.RNA沉默（RNA silencing），又称RNA干扰（RNA interference，RNAi），是一类调控真核生物基因表达的机制。小干扰RNA（small interfering RNAs，siRNA）与微RNA（microRNAs，miRNA）是多数真核生物中存在的两类主要RNAi相关小RNA（RNAi-associated small RNAs，smRNAs）。尽管多种古菌（Archaea）物种中存在大量长度超过50核苷酸（nucleotide，nt）的非编码RNA，但目前对古菌中类似真核生物20~24nt长smRNA的研究仍较为匮乏，这类smRNA已被证实参与基因的转录后表达调控。本研究基于深度测序（deep sequencing）技术，从超嗜热古菌硫磺矿硫化叶菌P2株（Sulfolobus solfataricus p2，Ssp2）中鉴定出大量长度约为20nt的smRNA，包括阶段性smRNAs（phased smRNAs）与潜在miRNA候选分子。我们证实了Ssp2中部分候选miRNA的表达。与Ssp2的超嗜热特性一致，研究发现：在以与Ssp2细胞提取物共孵育的推定回折型前体转录本（putative foldback precursor transcripts）构建的体外系统（in vitro system）中，更高温度可更高效地诱导候选miRNA的产生。尽管我们最初预测了部分候选miRNA的靶基因（target genes），但进一步分析显示，其切割位点位于候选miRNA互补区域的下游，这与植物miRNA介导的TAS转录本（TAS transcript）切割机制高度相似。我们还从成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeats，CRISPR）位点中鉴定出了smRNA，这类位点在原核微生物防御系统（prokaryotic microbial defense systems）中发挥关键作用。古菌是与细菌（Bacteria）、真核生物（Eukarya）并列的独特生命类群，本研究结果可为后续深入解析这类特殊生物中smRNA的调控机制与进化历程提供宝贵的研究资源。