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

Abstract

BackgroundMany 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.ResultsThrough 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.ConclusionsOur 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.

Protein-Coding cis-Natural Antisense Transcripts Have High and Broad Expression in Arabidopsis

“…Thus, one may expect that cellular sRNAs map to cis-NATs at high frequencies. Consistent with this idea, Zhang et al (2012) recently reported that siRNAs are overrepresented (P , 0.04) among the 39 UTRs of cis-NATs compared with 39 UTRs of non-cis-NATs (Zhang et al, 2012). In contrast, Henz et al (2007) found that sRNAs are more enriched in non-cis-NATs compared with cisNATs (Henz et al, 2007).…”

“…Across all genotypes, sRNAs matched PC non-cis-NATs from 1.4-to 5.2-fold more frequently than PC cis-NATs (Table IV). The frequency with which an sRNA sequenced from a putative nat-siRNA biogenesis mutant (dcl1, dcl2, dcl3, rdr2, rdr6; Borsani et al, 2005;Zhang et al, 2012) matched a PC cis-NAT was similar to the frequency with which an sRNA sequenced from wild-type plants and other sRNA mutants (dcl4, rdr1; Xie et al, 2004Xie et al, , 2005 matched PC cis-NATs (Table IV).…”

“…However, Zhang et al (2012), mapping a much larger number of unique sRNAs (17,141) to cis-NATs, reported that small interfering RNA (siRNA) target sites were overrepresented (P , 0.04) among the 39 untranslated regions (UTRs) of cis-NAT-encoding genes compared with 39 UTRs of non-cis-NAT-encoding genes (Zhang et al, 2012). We mapped sRNAs sequenced from wild-type and seven sRNA biogenesis mutant plants (Rajagopalan et al, 2006;Kasschau et al, 2007) and found that PC non-cis-NATs matched sRNAs more frequently than did PC cis-NATs across the eight genotypes (Supplemental Table S4).…”

“…More than 10,000 small RNAs (sRNAs) from plant stress sRNA libraries map to overlapping regions of cis-NAT transcripts (Zhang et al, 2012). Second, Jen et al, (2005) noted that cis-NAT-encoding genes had far more alternative splice variants and alternative polyadenylation sites than other genes, suggesting a function in antisense transcript-induced RNA splicing, alternative splicing, and polyadenylation.…”

Genome-wide profiling of sRNAs in the Verticillium dahliae-infected Arabidopsis roots

“…The genome loci of cis-NAT were referred to the previous study results (Jin et al 2008; Zhang et al 2012). Blast was used to match the sRNA to these loci.…”