Volume 6, Issue 3, Pages 830-846 (May 2013) A Global Identification and Analysis of Small Nucleolar RNAs and Possible Intermediate- Sized Non-Coding RNAs in Oryza sativa  Ting-Ting Liu, Danmeng Zhu, Wei Chen, Wei Deng, Hang He, Guangming He, Baoyan Bai, Yijun Qi, Runsheng Chen, Xing Wang Deng  Molecular Plant  Volume 6, Issue 3, Pages 830-846 (May 2013) DOI: 10.1093/mp/sss087 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Global characterization of rice non-coding RNAs (50–500 nt). (A) Functional distribution of ncRNAs. Rest: Observed ncRNAs of an unknown functional category. snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; SRP: signal recognition particle RNA; MRP: RNase mitochondrion RNA Processing RNA; NAT: natural antisense transcript; ITS: intergenic spacer; MIR: microRNA. (B) The number of classified ncRNA genes we identified compared with those found in public databases. (C) Venn diagram of differential expressed ncRNA genes in different stages. Stage I: mixed vegetative organs; Stage II; mixed reproductive organs. (D) Conservation analysis of ncRNAs from Rice, Arabidopsis, Sorghum and Brachypodium. Molecular Plant 2013 6, 830-846DOI: (10.1093/mp/sss087) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Genomic distribution of ncRNA genes in rice. (A) Chromosomal distribution of non-coding and protein-coding genes. (B) The distribution of ncRNA genes matched to the untranslated, coding, intronic and intergenic regions. Distribution of novel and classified ncRNAs around the transcription start site. (C) and 3’-end (D) of coding transcripts. Molecular Plant 2013 6, 830-846DOI: (10.1093/mp/sss087) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Gene organization of novel snoRNA gene clusters. (A) New clusters identified in this study. (B-C) Extended snoRNA clusters. (D) A novel snoRNA gene cluster partially antisense to an annotated protein-coding gene. (E) The proportion of snoRNAs found in singleton, tandem duplication and segmental duplication events. (F) The retention rate of intergenic and intragenic snoRNAs in segmental duplication blocks. White box: new snoRNA candidates; box with slash: annotated or predicted snoRNA genes in the public database; black box: exon regions of coding transcripts. Molecular Plant 2013 6, 830-846DOI: (10.1093/mp/sss087) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Relationship between gene duplication and multiple gene organizations. (A) Pie chart of all intronic snoRNAs with conserved and specific genomic organizations in Rice Sorghum and Brachypodium. Gene organizations of snoR104, snoR26a and their nested protein-coding genes in rice. (B, E), their counterparts in Sorghum (C, F), and Brachypodium (D,G). (H) Distance tree of the sequences of snoR134 gene variants in rice, Arabidopsis, Sorghum and Brachypodium. Gene organization of snoR111, snoR134 and their cluster containing variants in rice (I), Arabidopsis (J) and Sorghum (K) and Brachypodium(L) Molecular Plant 2013 6, 830-846DOI: (10.1093/mp/sss087) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Small RNAs derived from rice snoRNAs. (A) The size distribution of the sdRNAs. (B) Position-of-origin of the sdRNAs. (C) Predicted secondary structure of JNnc_loci1131. The red bar represents the position that the sdRNA is derived from. Total enriched ncRNAs (100 ug) were blotted, and the membranes were incubated with probes complementary to nt 9–57 (snoRNA probe) and to nt 2–23 (sdRNA probe) in (D) and (E). Molecular Plant 2013 6, 830-846DOI: (10.1093/mp/sss087) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 sdRNAs were primarily associated with AGO1b. (A) The major proportion of snoRNAs can produce sdRNA. (B) sdRNAs ranging from 19–21 nt were strongly enriched in AGO immunoprecipitates. (C) AGO proteins specifically enriched sdRNAs produced from 5’-end of snoRNAs (D) rice sdRNAs were strongly enriched in the AGO1b library. (E) 17 sdRNAs were enriched at least two fold in AGO1b immunoprecipitates. (F) sdRNAs with 5’ G and U were associated with rice AGO1b. (G) The relative enrichment of 47 selected sdRNA abundance in WT small RNA libraries compared to that observed in DCL1IR-2 (blue) and dcl3a-17 (red) RNAi libraries, respectively. snoRNAs were employed when the normalized reads of their sdRNAs were more than 5 in each library and the relative enrichment of abundance were at least more than 2-fold. Molecular Plant 2013 6, 830-846DOI: (10.1093/mp/sss087) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions