Unexpected diversity of chloroplast non-coding RNAs as revealed by deep sequencing of the Arabidopsis transcriptome. Arabidopsis thaliana

Non-coding RNAs (ncRNAs) are widely expressed in both prokaryotes and eukaryotes. Eukaryotic ncRNAs are commonly small (18-25 nt) micro- and small interfering RNAs involved in post-transcriptional gene silencing, while prokaryotic ncRNAs vary in size and are involved in various aspects of gene regulation. Given the prokaryotic origin of organelles, the presence of ncRNAs might be expected, however, the full spectrum of organellar ncRNAs has not been determined systematically. Here, strand-specific RNA-Seq analysis was used to identify 107 candidate ncRNAs from Arabidopsis thaliana chloroplasts, primarily encoded opposite protein- coding and tRNA genes. Forty-eight ncRNAs were shown to accumulate by RNA gel blot as discrete transcripts in wild-type (WT) plants and/or the pnp1-1 mutant, which lacks the chloroplast ribonuclease polynucleotide phosphorylase (cpPNPase). Ninety-eight percent of the ncRNAs detected by RNA gel blot had different transcript patterns between WT and pnp1-1, suggesting cpPNPase has a significant role in chloroplast ncRNA biogenesis and accumulation. Analysis of materials deficient for other major chloroplast ribonucleases, RNase R, RNase E and RNase J, showed differential effects on ncRNA accumulation and/or form, suggesting specificity in RNase-ncRNA interactions. 5' end mapping showed that some ncRNAs are transcribed from dedicated promoters, while others result from transcriptional read-through. Finally, correlations between accumulation of some ncRNAs and the symmetrically-transcribed sense RNA are consistent with a role in RNA stability. Overall, our data suggest that this extensive population of ncRNAs has the potential to underpin a previously underappreciated regulatory mode in the chloroplast.

非编码RNA（non-coding RNAs，ncRNAs）广泛分布于原核生物（prokaryotes）与真核生物（eukaryotes）中。真核生物来源的非编码RNA通常为长度18~25 nt的微小RNA及小干扰RNA，参与转录后基因沉默（post-transcriptional gene silencing）过程；原核生物的非编码RNA长度各异，参与基因调控的多个环节。鉴于细胞器起源于原核生物，理论上细胞器中应存在非编码RNA，但目前尚未有系统性研究明确细胞器非编码RNA的完整谱图。本研究采用链特异性RNA测序（RNA-Seq）技术，从拟南芥（Arabidopsis thaliana）叶绿体中鉴定得到107个候选非编码RNA，这些RNA主要编码于蛋白质编码基因与转运RNA（transfer RNA，tRNA）基因的反义链上。经RNA印迹杂交（RNA gel blot）验证，48个非编码RNA在野生型（wild-type，WT）植株及/或缺失叶绿体核糖核酸酶多核苷酸磷酸化酶（chloroplast ribonuclease polynucleotide phosphorylase，cpPNPase）的pnp1-1突变体中以离散转录本的形式积累。在RNA印迹杂交检测到的非编码RNA中，98%在野生型与pnp1-1突变体中呈现出不同的转录本模式，这表明cpPNPase在叶绿体非编码RNA的生成与积累过程中发挥重要作用。对其他主要叶绿体核糖核酸酶（RNase R、RNase E及RNase J）缺失材料的分析显示，这些酶对非编码RNA的积累和/或形态具有差异化调控作用，提示核糖核酸酶与非编码RNA之间存在特异性相互作用。5'端定位分析表明，部分非编码RNA由特异性启动子转录而来，而另一些则源于转录通读（transcriptional read-through）事件。最后，部分非编码RNA的积累水平与其对称转录的正义RNA之间存在相关性，这提示其可能参与RNA稳定性调控。综合来看，本研究数据表明，这类丰富的非编码RNA群体可能支撑了叶绿体中此前未被充分认知的调控模式。