Genome-wide analysis of plant nat-siRNAs reveals insights into their distribution, biogenesis and function. Genome Biol 13(3):R20

Department of Plant Pathology and Microbiology, Center for Plant Cell Biology, University of California, Riverside, CA 92521, USA.
Genome biology (Impact Factor: 10.81). 03/2012; 13(3):R20. DOI: 10.1186/gb-2012-13-3-r20
Source: PubMed


Many eukaryotic genomes encode cis-natural antisense transcripts (cis-NATs). Sense and antisense transcripts may form double-stranded RNAs that are processed by the RNA interference machinery into small interfering RNAs (siRNAs). A few so-called nat-siRNAs have been reported in plants, mammals, Drosophila, and yeasts. However, many questions remain regarding the features and biogenesis of nat-siRNAs.
Through deep sequencing, we identified more than 17,000 unique siRNAs corresponding to cis-NATs from biotic and abiotic stress-challenged Arabidopsis thaliana and 56,000 from abiotic stress-treated rice. These siRNAs were enriched in the overlapping regions of NATs and exhibited either site-specific or distributed patterns, often with strand bias. Out of 1,439 and 767 cis-NAT pairs identified in Arabidopsis and rice, respectively, 84 and 119 could generate at least 10 siRNAs per million reads from the overlapping regions. Among them, 16 cis-NAT pairs from Arabidopsis and 34 from rice gave rise to nat-siRNAs exclusively in the overlap regions. Genetic analysis showed that the overlapping double-stranded RNAs could be processed by Dicer-like 1 (DCL1) and/or DCL3. The DCL3-dependent nat-siRNAs were also dependent on RNA-dependent RNA polymerase 2 (RDR2) and plant-specific RNA polymerase IV (PolIV), whereas only a fraction of DCL1-dependent nat-siRNAs was RDR- and PolIV-dependent. Furthermore, the levels of some nat-siRNAs were regulated by specific biotic or abiotic stress conditions in Arabidopsis and rice.
Our results suggest that nat-siRNAs display distinct distribution patterns and are generated by DCL1 and/or DCL3. Our analysis further supported the existence of nat-siRNAs in plants and advanced our understanding of their characteristics.

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    • "Among the 135 cis-NATs in Cassava, 48 (36%), 15 (11%) and 72 (53%) pairs were arranged in the convergent (3′-3′ overlap), divergent (5′-5′ overlap) and enclosed orientations, respectively (Additional file 2: Tables S9). Note that the percentage of enclosed cis-NATs was greater than that in Arabidopsis [25, 26]. In castor bean, the same number of 23 cis-NAT pairs appeared in the enclosed and divergent categories and 17 were convergent (Additional file 2: Table S10). "
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    ABSTRACT: Small noncoding RNA (sncRNA), including microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs) are key gene regulators in eukaryotes, playing critical roles in plant development and stress tolerance. Trans-acting siRNAs (ta-siRNAs), which are secondary siRNAs triggered by miRNAs, and siRNAs from natural antisense transcripts (nat-siRNAs) are two well-studied classes of endo-siRNAs. In order to understand sncRNAs’ roles in plant chilling response and stress acclimation, we performed a comprehensive study of miRNAs and endo-siRNAs in Cassava (Manihot esculenta), a major source of food for the world populations in tropical regions. Combining Next-Generation sequencing and computational and experimental analyses, we profiled and characterized sncRNA species and mRNA genes from the plants that experienced severe and moderate chilling stresses, that underwent further severe chilling stress after chilling acclimation at moderate stress, and that grew under the normal condition. We also included castor bean (Ricinus communis) in our study to understand conservation of sncRNAs. In addition to known miRNAs, we identified 32 (22 and 10) novel miRNAs as well as 47 (26 and 21) putative secondary siRNA-yielding and 8 (7 and 1) nat-siRNA-yielding candidate loci in Cassava and castor bean, respectively. Among the expressed sncRNAs, 114 miRNAs, 12 ta-siRNAs and 2 nat-siRNAs showed significant expression changes under chilling stresses. Systematic and computational analysis of microRNAome and experimental validation collectively showed that miRNAs, ta-siRNAs, and possibly nat-siRNAs play important roles in chilling response and chilling acclimation in Cassava by regulating stress-related pathways, e.g. Auxin signal transduction. The conservation of these sncRNA might shed lights on the role of sncRNA-mediated pathways affected by chilling stress and stress acclimation in Euphorbiaceous plants.
    Full-text · Article · Jul 2014 · BMC Genomics
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    • "Another class of siRNAs is nat-siRNAs, which could be derived from RNAs transcribed from opposite strands of the same loci (cis-nat-siRNAs) [12] or by transcripts from different loci (trans-nat-siRNAs). There are 1,739 and 4,828 potential cis- and trans- natural antisense transcripts (NATs), respectively in Arabidopsis[13]. The production of nat-siRNAs are is dependent on RDR6 and DCL2 (24-nt) or DCL1 (21-nt). "
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    ABSTRACT: In higher eukaryotes, small RNAs play a role in regulating gene expression. Overexpression (OE) lines of Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) were shown to grow faster and exhibit higher ATP and sugar contents. Leaf microarray studies showed that many genes involved in microRNAs (miRNAs) and trans-acting siRNAs (tasiRNAs) biogenesis were significantly changed in the fast-growing lines. In this study, the sRNA profiles of the leaf and the root of 20-day-old plants were sequenced and the impacts of high energy status on sRNA expression were analyzed. 9-13 million reads from each library were mapped to genome. miRNAs, tasiRNAs and natural antisense transcripts-generated small interfering RNAs (natsiRNAs) were identified and compared between libraries. In the leaf of OE lines, 15 known miRNAs increased in abundance and 9 miRNAs decreased in abundance, whereas in the root of OE lines, 2 known miRNAs increased in abundance and 9 miRNAs decreased in abundance. miRNAs with increased abundance in the leaf and root samples of both OE lines (miR158b and miR172a/b) were predicted to target mRNAs coding for Dof zinc finger protein and Apetala 2 (AP2) proteins, respectively. Furthermore, a significant change in the miR173-tasiRNAs-PPR/TPR network was observed in the leaves of both OE lines. In this study, the impact of high energy content on the sRNA profiles of Arabidopsis is reported. While the abundance of many stress-induced miRNAs is unaltered, the abundance of some miRNAs related to plant growth and development (miR172 and miR319) is elevated in the fast-growing lines. An induction of miR173-tasiRNAs-PPR/TPR network was also observed in the OE lines. In contrast, only few cis- and trans-natsiRNAs are altered in the fast-growing lines.
    Full-text · Article · Feb 2014 · BMC Genomics
    • "More than 1,000 pairs of NAT were predicted in the rice genome (Lu et al. 2012). It is well known that the NAT can produce small RNAs through the siRNA biogenesis pathway (Borsani et al. 2005; Zhang et al. 2012). The factors determining the abundance of MITE-derived small RNAs remain unclear. "
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    ABSTRACT: More and more evidence has accumulated in the past 20 years suggesting that MITEs may have played important roles in plant gene and genome evolution. With a large number of plant genomes sequenced and the development of computational programs for de novo MITE identification, a massive number of MITEs have been identified from plant genomes. The number of MITEs in a genome varied dramatically among different plant species. There is significant correlation between the number of MITEs and genome size, though there are several prominent exceptions. Some MITE families have a high copy number in a genome, probably due to one or several rounds of amplification bursts. Different MITE families in the same genome may have experienced amplification burst at different times, suggesting that their amplifications were triggered by distinct environments (such as stress) or genetic events. However, very few MITEs in plant genomes are currently active. MITEs are often distributed in gene-rich regions, and may be inserted in genes’ promoter regions or transcribed regions. They may affect (either upregulate or downregulate) the expression of nearby genes. MITEs may downregulate genes through small RNAs, which may be produced via NAT or double-stranded RNAs formed by transcribed MITE sequences. The presence/absence of MITEs as well as their potential effects on expression of nearby genes suggests that MITE may provide considerable physiological and phenotypic variations for a species. Important future studies on MITEs include the mechanisms of MITE activation and the effects of MITEs on gene and genome evolution.
    No preview · Chapter · Jan 2014
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