Wang et al. BMC Genomics 2011, 12:289 http://www.biomedcentral.com/1471-2164/12/289 Table 2 Heat response of tRNA-derived csRNAs Group I II III IV Ala,Asp,Phe,Lys,Leu,Gln,Thr,Val Cys,Glu,Ile,Met,Asn,Pro,Ser,Trp tRNA His,Arg Gly Heat response Shorter csRNAs increased, and longer csRNAs decreased b Shorter csRNAs increased, and longer csRNAs unchanged Longer csRNAs decreased, and shorter csRNAs unchanged 18 nt csRNAs decreased, and the others unchanged a: Percentage for each group in total abundance of tRNA-derived csRNAs. b: The shorter csRNAs ranged from 15 to 25 nt in length, and the longer csRNAs were from 29 to 32 nt. Page 8 of 14 Percentage (%) a MT HT 81 12 5 2 73 20 5 2 Figure 7 Identification of csR-trnA-1 and csR-trnA-2. (A) An diagram showing the location of csR-trnA-1 and csR-trnA-2 in chloroplast tRNA-Ala. Two exons of tRNA-A are indicated by boxes. (B) and (C) Size distribution of the csR-trnA-1 family and the csR- trnA-2 family in sequence datasets with core sequences (underlined in Additional File 10). (D) and (E) Northern blotting of csR-trnA-1 and csR-trnA-2 in the HT seedlings of Chinese cabbage. U6 spliceosomal RNA was used as an RNA loading control. Putative structure of csRNAs and their precursors To associate small RNAs with the folded conformations of RNA, we analyzed the secondary structure of some rRNA and tRNA molecules. According to the secondary structures of 5S and 4.5S rRNA from E. coli [31,32] and 16S rRNA from Haloferax volcanii [33], we simulated the secondary structure of some rRNAs-csRNAs of Chi- nese cabbage with high abundance. csR-5sr-1 and csR- 5sr-2, two of the major csRNA sets derived from the Chinese cabbage chloroplast 5S rRNA (Additional File 9), matched a “single-stem-with-bulge” frame (type I) and a “self-hairpin” frame (type II) of 5S rRNA (Figure 9A). The parallel analysis of the csRNAs from 4.5S RNA of E. coli indicated that two csRNAs derived from 4.5S rRNA and three csRNAs derived from 16S RNA were located in “single-stem-with-bulge” frame (Additional File 11A). Meanwhile, three of the other csRNAs from 16S rRNA present a potential secondary structure of “self-hairpin” (Additional File 9). csR-16sr-1 is the most abundant csRNA from 16S rRNA. This csRNA and csR- 16sr-2 exhibited “two-stem-with-hinge” frame (Addi- tional File 11B). tRNA-derived small RNAs were mostly identified by starting from the first nucleotide of the mature tRNA sequences. Compared to the cloverleaf structures of the tRNA, this position-specificity resulted in a 25 nt csRNA that had the exact same sequence as the accep- tor arm from the 5’ end, including the D-arm and D-loop of the tRNA; a few of nucleotides of csRNAs of 27~31 nt were matched to the stem of anticodon arm; and even a 36 nt csRNA was paired with the complete anticodon (Additional File 11C). Zhang et al. [27] reported a class of ~58-60 nt non-coding RNAs in the phloem sap of pumpkin, whose cleavage sites fell in the anticodon and D arm of tRNA. csRNAs of Chinese cab- bage are much shorter than these non-coding RNAs, but both are generated in anticodon or D loop of tRNA. To investigate the conceivable precursors of the csRNA sequences, circular RT-PCR was carried out. Two different RNA fragments of csR-5sr-1 precursor were isolated and sequenced (Figure 9B). Pre-1 was 141 nt in length, 20 nt longer than mature sequence of 5S rRNA, with a 3 nt extension to the 5’ ends, and a 15 nt