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

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Distinct distribution patterns of Arabidopsis nat-siRNAs. (a-i) Arabidopsis nat-siRNAs displaying a distributed pattern and exclusively derived from the overlapping regions. (j-o) Arabidopsis nat-siRNAs displaying a distributed pattern but derived from both the overlap region and non-overlap region. sRNAs matching the upper and lower strands are displayed above and below the NAT pairs, respectively. The red regions on the gene model represent exons, whereas the blue regions represent introns. The region between the green lines represents the overlapping region of the NATs. siRNAs probed are indicated by black arrows.

Figure 2

Distribution of rice nat-siRNAs with distribution pattern OS03G02762/OS03G02770 (a) and OS08G06220/OS08G06230 (b) or site-specific pattern OS04G57140/OS04G57160 (c). Strands above or under the NAT pairs represent sRNAs positively or negatively mapping to the upper strands. In the gene model, exons and introns are indicated by red regions and blue regions, respectively. The overlapping regions of the NATs are indicated by green lines.

Figure 3

Site-specific patterns of Arabidopsis nat-siRNAs At4g35850/At4g35860 (a), At2g33793/At2g33796 (b), and At1g51400/At1g51402 (c). siRNAs that positively or negatively map to the upper genes are present above or under the gene pair, respectively. The introns of cis-NATs are represented by blue strands and overlapping regions of cis-NATs are shown by green lines. The black arrow represents the siRNAs detected by Northern blot.

Figure 4

Biogenesis of nat-siRNAs that depend on DCL1. (a-c) Accumulation of nat-siRNA1g51400 (a), nat-siRNA2g41590 (b) and nat-lsiRNA4g34070 (c) is shown in various dcl, rdr, polIV and polV mutants. Total RNA (75 to 100 μg) was used for Northern blot analysis. LNA probes complementary to each nat-siRNA were used. U6 was an internal control to show equal loading. The same results were obtained from two biological replicates.

Figure 5

Biogenesis of nat-siRNAs that depend on DCL3. (a-c) Accumulation of nat-siRNA1g13340 (a), nat-siRNA1g28100 (b) and nat-siRNA1g17745 (c)Total RNA (75 to 100 μg) of distinct dcl, rdr, polIV and polV mutants and corresponding WT are probed by LNA probes indicated in Figures 1 and 3. U6 was used as a loading control. Similar results were obtained in two biological repeats.

Figure 6

Expression of the NAT transcripts is increased in dcl1-7 fwf2 and dcl3-1 mutants. Expression was examined by quantitative RT-PCR and Actin2 was used as an internal control. Total RNA (5 μg) was treated with DNaseI and then subjected to reverse transcription. Error bars indicate standard deviations derived from three technical replicates. Similar results were obtained from two biological replicates.