G3 (Bethesda). 2011 Dec;1(7):559-70. doi: 10.1534/g3.111.000752. Epub 2011 Dec 1.
Noncoding RNAs (ncRNA) are widely expressed in both prokaryotes and eukaryotes. Eukaryotic ncRNAs are commonly micro- and small-interfering RNAs (18-25 nt) involved in posttranscriptional gene silencing, whereas 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 demonstrates that some ncRNAs are transcribed from dedicated promoters, whereas 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（ncRNA）广泛存在于原核生物和真核生物中。真核 ncRNA 通常是 18-25 个核苷酸的 micro 和 small-interfering RNA（miRNA 和 siRNA），参与转录后基因沉默，而原核 ncRNA 大小不一，参与基因调控的各个方面。鉴于细胞器具有原核起源，ncRNA 的存在是可以预期的；然而，尚未系统地确定细胞器 ncRNA 的全部范围。在这里，使用链特异性 RNA-Seq 分析从拟南芥叶绿体中鉴定了 107 个候选 ncRNA，主要由编码蛋白和 tRNA 的基因的反义编码。在野生型（WT）植物和/或缺乏叶绿体核糖核酸酶多核苷酸磷酸化酶（cpPNPase）的 pnp1-1 突变体中，有 48 个 ncRNA 被 RNA 凝胶印迹作为离散转录本积累。在 WT 和 pnp1-1 之间，通过 RNA 凝胶印迹检测到的 98%的 ncRNA 具有不同的转录模式，这表明 cpPNPase 在叶绿体 ncRNA 生物发生和积累中具有重要作用。对其他主要叶绿体核糖核酸酶，RNase R、RNase E 和 RNase J 缺乏的材料进行分析表明，ncRNA 积累和/或形式存在差异效应，这表明 RNase-ncRNA 相互作用具有特异性。5' 端映射表明，一些 ncRNA 是由专用启动子转录的，而其他 ncRNA 则是转录通读的结果。最后，一些 ncRNA 积累与对称转录的有义 RNA 之间的相关性与 RNA 稳定性相关。总体而言，我们的数据表明，这一广泛的 ncRNA 群体有可能为叶绿体中以前被低估的调控模式提供支持。
