Wang et al. BMC Genomics 2011, 12:289 http://www.biomedcentral.com/1471-2164/12/289 Page 10 of 14 Figure 9 The predicted secondary structures and putative precursors of csR-5sr-1 and csR-5sr-2. (A) The prediction of csR- 5sr-1 and csR-5sr-2 on the secondary structure of 5S rRNA from E. coli, according to their location numbers on Chinese cabbage chloroplast 5S rRNA. (B) The sequenced transcripts of putative precursor of csR-5sr-1. The dashed line indicates the internal deletion. (C) Alignment of the putative precursors of csR-5sr-1 with 5S rDNA. The arrow and round-head arrow directed the first and last nucleotides of 5S rRNA, respectively. csR-5sr-1 sequence is underlined in red color. The dotted lines indicate the position of PCR primer 2. possible targets paired with csR-RPO-1 and csR-mPSB-6 csRNAs, respectively (Additional File 9). As shown by small RNA deep-sequencing and microRNA microarray, these two csRNAs were remarkably increased by heat stress in the HT seedlings (Figure 10A). Opposite to this, the expression of the rpoA and psbM gene was down-regulated in the HT seedlings (Figure 10B). These consistent results revealed that csRNAs play certain roles in regulation of gene expression. Discussion Chloroplast RNA generates a novel subset of small RNAs Although studies in recent years have reported the exis- tence of small non-coding RNAs in DNA-containing cellular organelles [10-12], a genome-wide chloroplast profiling study is not reported yet, and therefore, the expression patterns, biological functions and biogenesis pathways of stable small RNA species remain largely unknown. From small RNA libraries of both Chinese cabbage and Arabidopsis, we selected two classes of the Figure 10 Expression analysis of the four putative target genes of csRNAs. (A) A csRNA abundance comparison between HT and MT samples from Chinese cabbage deep-sequencing datasets. (B) Fold changes of putative target gene expression. chloroplast-related small RNAs (csRNAs). By comparing chloroplast-specific small RNAs with csRNAs in Arabi- dopsis, we showed that most of the small RNAs in the csRNA dataset were derived exclusively from the chloro- plast genome rather than the nuclear or mitochondrial genomes; thus, csRNA dataset may represent the fea- tures of chloroplast-derived small RNAs. Using highly purified chloroplast RNA samples, we confirmed by Northern blotting that most of the csRNAs were pro- duced and accumulated inside chloroplasts. Among csRNAs from the ~150 kb sequence of the chloroplast genome, about 90% were mapped to chloro- plast rRNA and tRNA loci. Although fragments from rRNA and tRNA are regularly considered to be degrada- tion contaminants, we present several clues suggesting a different viewpoint: a) Most of the tRNA/rRNA-derived csRNAs were associated with the 5’ ends of tRNAs or 3’ ends of rRNAs, respectively, and were even precisely aligned to the first nucleotides of mature tRNA,