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Genome-wide analysis of plant nat-siRNAs reveals insights into their distribution, biogenesis and function

Xiaoming Zhang1, Jing Xia2, Yifan E Lii1, Blanca E Barrera-Figueroa34, Xuefeng Zhou2, Shang Gao1, Lu Lu15, Dongdong Niu16, Zheng Chen2, Christy Leung1, Timothy Wong1, Huiming Zhang7, Jianhua Guo16, Yi Li5, Renyi Liu3, Wanqi Liang8, Jian-Kang Zhu7, Weixiong Zhang29* and Hailing Jin1*

Author Affiliations

1 Department of Plant Pathology and Microbiology, Center for Plant Cell Biology and Institute for Integrative Genome Biology, University of California, Riverside, CA 92521, USA

2 Department of Computer Science and Engineering, Washington University in St Louis, St Louis, MO 63130, USA

3 Department of Botany and Plant Sciences, Center for Plant Cell Biology and Institute for Integrative Genome Biology, University of California, Riverside, CA 92521, USA

4 Instituto de Biotecnologia, Universidad del Papaloapan, Tuxtepec Oaxaca 68301, Mexico

5 Peking-Yale Joint Center for Plant Molecular Genetics and Agrobiotechnology, The State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, 100871, China

6 Department of Plant Protection, Nanjing Agriculture University, Nanjing, 210095, China

7 Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA

8 School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China

9 Department of Genetics, Washington University School of Medicine, St Louis, MO 63110, USA

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Genome Biology 2012, 13:R20  doi:10.1186/gb-2012-13-3-r20

Published: 22 March 2012

Additional files

Additional file 1:

Distributions of the sequencing reads from small RNA libraries of abiotic and biotic treated Arabidopsis and abiotic challenged rice. Shown in the table are the total number of raw sequencing reads (total), the number of qualified reads that can map perfectly to the corresponding Arabidopsis or rice genome (mapped), intergenic regions (intergenic), intronic sequences (introns), transposons or repeats (repeats/mobile), tRNA, rRNA, snoRNA and snRNA sequences (ncRNA), trans-acting siRNA (ta-siRNA), microRNA sequences (miRNAs), intron-exon junctions (intron-exon junction) and exon-exon junctions (exon-exon junction). No mismatches were allowed in the mapping. The second number for each condition (column) is the percentage of reads relative to the total mapped reads.

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Additional file 2:

(a-d) Distributions of the lengths (a, b) and the first nucleotides (c, d) of total siRNAs and nat-siRNAs in stress-challenged Arabidopsis and rice. (a) Length distributions of unique sequencing reads in Arabidopsis. The blue and red bars represent total siRNAs and nat-siRNAs, respectively. (b) Length distributions of unique sequencing reads in rice. The blue and red bars represent total siRNAs and nat-siRNAs, respectively. (c) First-nucleotide distribution of unique sequencing reads in Arabidopsis. (d) First-nucleotide distribution of unique sequencing reads in rice.

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Additional file 3:

Normalized reads of siRNAs mapped to the overlapping regions of Arabidopsis and rice cis-NAT pairs, respectively (a) siRNAs mapped to the overlapping regions of 84 Arabidopsis cis-NAT pairs. (b) siRNAs mapped to the overlapping regions of 119 rice cis-NAT pairs. Each small RNA library was normalized to one million reads with 100% matching to the genome. The sum of normalized reads from each library was listed for each cis-NAT pair.

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Additional file 4:

Arabidopsis cis-NATs with two classes of small RNAs mapped to the overlapping regions. 'gene1' and 'gene2' represent the first and second transcript in each cis-NAT pair. Listed are 84 Arabidopsis cis-NATs with siRNAs mapped to the overlapping region. Raw reads of 20- to 22-nucleotide and 23- to 28-nucleotide classes of siRNAs matching 100% to the genome were analyzed. The percentage of 20- to 22-nucleotide and 23- to 28-nucleotide siRNAs are presented.

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Additional file 5:

(a) Distributions of the number of small RNA clusters in 84 Arabidopsis cis-NATs. The red line represents the separation between site-specific (left) and distributed (right) patterns. (b) The plot of two metrics of 84 cis-NATs in Arabidopsis. Each dot represents the number of clusters within the cis-NAT whole region and the percentage of small RNA reads in all clusters. The red dots in the rectangle were classified as site-specific patterns, whereas the blue dots were distributed patterns, represented by a linear correlated line.

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Additional file 6:

Classification of cis-NAT pairs. We analyzed 84 Arabidopsis and 119 rice cis-NAT pairs that have more than 10 raw siRNAs mapped to the overlap region. The reads analyzed here are the raw sequencing reads mapped to the whole region from the combination of all libraries. (a) Arabidopsis cis-NAT pairs display different distribution patterns. (b) Rice cis-NAT pairs display different distribution patterns.

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Additional file 7:

Oligos used in this study. A plus sign ('+') before the nucleotide depicts LNA residues.

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Additional file 8:

Small RNAs mapped to the introns or intron-exon junction regions of Arabidopsis cis-NATs. Copy number indicates the number of raw reads in the combination of all libraries. (a) siRNAs mapping to introns in the overlapping region of cis-NATs in Arabidopsis. (b) siRNAs mapping to introns in the whole region of cis-NATs in Arabidopsis.

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Additional file 9:

Small RNAs mapped to the introns or intron-exon junction regions of rice cis-NATs. Raw reads from all the libraries were analyzed. (a) siRNAs mapping to introns in the overlapping region of cis-NATs in rice. (b) siRNAs mapping to introns in the whole region of cis-NATs in rice

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Additional file 10:

Normalized reads of siRNAs mapped to 84 Arabidopsis NATs under different conditions. Each library was normalized to a million reads that perfectly mapped to the genome. The fold change with maximum absolute value between treated sample and corresponding control is listed as a supplemental value. The pairs with more than two-fold change were annotated in red (up) or in blue (down). (a) Reads of small RNAs mapped to overlapping region of NATs under different conditions. (b) Reads of small RNAs mapped to whole region of NAT under different conditions.

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Additional file 11:

Normalized reads of siRNAs mapped to 119 rice NATs under different conditions. siRNAs matching 100% to the genome were analyzed and each library was normalized to a million reads before analyzing. The fold change with maximum absolute value between treated and untreated libraries is listed. The pairs with more than a two-fold change are displayed in red (up, +) or blue (down, -). (a) Reads of small RNAs mapped to overlapping region of NATs under different conditions. (b) Reads of small RNAs mapped to whole region of NATs under different conditions.

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Additional file 12:

Arabidopsis cis-NATs that are up-regulated in the dcl1-7 mutant. (a) List of probes of the 93 genes of the 84 cis-NAT pairs. Annotations of the Affymetrix ATH1-121501 chip were used and no probes were found for the rest of the cis-NAT genes. (b) The 23 cis-NAT genes up-regulated in dcl1-7.

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Additional file 13:

Expression analysis of NAT transcripts in the fwf2 single mutant. The expression of NAT transcripts was analyzed by quantitative RT-PCR. Total RNA (5 μg) was used for DNase treatment and reverse transcription. Error bars indicate the technical replicates and similar results were obtained from two biological repeats.

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Additional file 14:

Association of 20- to 22-nucleotide and 23- to 26-nucleotide classes of nat-siRNAs with different AGOs in Arabidopsis. Unique reads of nat-siRNAs associated with AGO1, AGO2, AGO4 and AGO7 were analyzed. Loaded reads represent unique reads of nat-siRNAs that were loaded into distinct AGOs. The percentage in total nat-siRNA-AGO libraries indicates the distribution of loaded nat-siRNAs in different AGOs.

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Additional file 15:

SRO5-P5CDH siRNAs derived from salt and cold stress challenged Arabidopsis. The siRNAs were identified from GEO database accession number GSE33642. (a) Reads of siRNAs positively/negatively match to At5G62520 (SRO5) in the overlap/non-overlap region. The '+ strand' and '- strand' indicate positively or negatively matching. (b) Distribution pattern of SRO5-P5DCH siRNAs. siRNAs positively or negatively matched to At5G62520 are displayed above or below the SRO5-P5DCH gene pair. Exons and introns are represented by red and blue dishes, respectively. The overlapping region is indicated by the two green lines.

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