Role of transposon-derived small RNAs in the interplay between genomes and parasitic DNA in rice

Abstract

RNA silencing is a defense system against "genomic parasites" such as transposable elements (TE), which are potentially harmful to host genomes. In plants, transcripts from TEs induce production of double-stranded RNAs (dsRNAs) and are processed into small RNAs (small interfering RNAs, siRNAs) that suppress TEs by RNA-directed DNA methylation. Thus, the majority of TEs are epigenetically silenced. On the other hand, most of the eukaryotic genome is composed of TEs and their remnants, suggesting that TEs have evolved countermeasures against host-mediated silencing. Under some circumstances, TEs can become active and increase in copy number. Knowledge is accumulating on the mechanisms of TE silencing by the host; however, the mechanisms by which TEs counteract silencing are poorly understood. Here, we show that a class of TEs in rice produces a microRNA (miRNA) to suppress host silencing. Members of the microRNA820 (miR820) gene family are located within CACTA DNA transposons in rice and target a de novo DNA methyltransferase gene, OsDRM2, one of the components of epigenetic silencing. We confirmed that miR820 negatively regulates the expression of OsDRM2. In addition, we found that expression levels of various TEs are increased quite sensitively in response to decreased OsDRM2 expression and DNA methylation at TE loci. Furthermore, we found that the nucleotide sequence of miR820 and its recognition site within the target gene in some Oryza species have co-evolved to maintain their base-pairing ability. The co-evolution of these sequences provides evidence for the functionality of this regulation. Our results demonstrate how parasitic elements in the genome escape the host's defense machinery. Furthermore, our analysis of the regulation of OsDRM2 by miR820 sheds light on the action of transposon-derived small RNAs, not only as a defense mechanism for host genomes but also as a regulator of interactions between hosts and their parasitic elements.

Figure 1. miR820 family members are located within CACTA transposons and target the DNA methyltransferase gene OsDRM2.

(A) The location of miR820 within the CACTA DNA transposon is shown in the first row. The second and third rows indicate the similarity between the sequences of the miR820 precursor (pre-miR820) and Os03g0110800 (OsDRM2). The numbers beside the lines indicate the nucleotide identities between the regions. The red triangle indicates the location of miR820 within the stem-loop region. (B) Northern blot analysis of miR820 expression in the wild-type (WT) and in waf1 mutants. (C) Detection of miR820-cleaved OsDRM2 mRNA by RNA ligation–mediated 5′ RACE (upper panel). The same cDNA templates were used for PCR to amplify OsDRM2 (middle panel) and OsActin (bottom panel) as controls for OsDRM2 expression and RNA integrity. (D) qRT-PCR analysis measuring the expression level of OsDRM2 in waf1-1, waf1-2, and WT. The expression level of the WT was set as 1. Values are means, with bars showing standard errors. Significance was assessed by a two-tailed Student’s t-test; (**) significant at the 1% level; (*) significant at the 5% level. The n value represents the number of mutant or wild-type individuals.

Figure 2. OsDRM2 is negatively regulated by miR820.

(A) The general structure of the OsDRM2::GFP fusion constructs is shown at the bottom, and the sequences of miR820a/b/c and the target sites in the 35S:OsDRM2 intact:GFP, 35S:OsDRM2 mutation1:GFP, and 35S:OsDRM2 mutation2:GFP constructs are shown at the top. The changed nucleotides in the mutant genes are shown in red letters. (B) The panels on the left show GFP fluorescence and white-light observations of longitudinal sections of shoots of transgenic plants transformed with the constructs in (A). The graph at the right shows relative expression levels of GFP mRNA in the corresponding transgenic lines as measured by quantitative RT-PCR. The expression level of OsDRM2-intact lines was set as 1. (C) Relative expression levels of OsDRM2 and TEs in OsDRM2 RNAi transgenic callus (black bars) measured by qRT-PCR. The expression level of empty-vector lines was set as 1. (D) McrBC-PCR analysis of genomic DNA from callus of WT and two independent transgenic lines of OsDRM2 RNAi. Two of the six OsDRM2 RNAi transgenic lines analyzed in (C) were used. In (B) and (C), values are means, with bars showing standard errors. Significance was assessed by a two-tailed Student's t-test; (**) significant at the 1% level; (*) significant at the 5% level. The n value represents the number of independent transformants.

Figure 3. Regulation of DRM2 by miR820 is conserved among Oryza species.

(A) The phylogenetic tree shows the evolutionary relationships between Oryza species (left). Boxes indicate the types of miR820 sequences and DRM2 target site sequences identified in each genome (right). Within each column, boxes of the same color indicate identical sequences. The genome origins of the sequences in the boxes are indicated by arrows. (B) Sequence alignments of miR820 and its target site in DRM2 in the AA (Nipponbare; blue box) and BB/BBCC genomes (pink box). Dots between nucleotides indicate the type of nucleotide pair: a double dot indicates an A-U or G-C pair, a single dot indicates a G-U pair, and no dot indicates a mismatch. The positions of G-U pairs and mismatches are shown with broken and solid arrows, respectively. The eight nucleotide substitutions found between AA and BB/BBCC Oryza species are highlighted in yellow. Blue lines above and below the sequence of DRM2 indicate the codons. Letters above and below the lines indicate the amino acids encoded by DRM2 in AA and BB/BBCC Oryza species, respectively. Phylogenetic tree in (A) adapted from Ge et al. (1999) .

Figure 4. Increased copy number of CACTA carrying pre-miR820 in the BB/BBCC genome.

(A) Detection of CACTA TEs carrying pre-miR820 by Southern blot analysis. Genomic DNA from AA, BB, BBCC, and CC Oryza species were digested with the enzymes indicated and probed with pre-miR820. Red triangles indicate the bands corresponding to the five copies of pre-miR820 in Nipponbare. The number next to each triangle indicates the chromosome location of that copy. (B) Mapping of CACTA carrying pre-miR820 in the rice genome. The genomic locations of CACTA carrying pre-miR820 in AA and BB Oryza species are shown by gray arrows and black arrows, respectively. (C) Phylogenetic analysis of miR820-CACTA sequences. Bootstrap values (1000 replicates) are given for branch nodes. Black and red numbers in parentheses indicate the chromosome locations of pre-miR820 sequences in the AA and BB genomes, respectively. Multiple copies on one branch, indicating that the identical sequence was found at multiple loci, are highlighted in blue. (D) A model for the regulation of DRM2 by miR820. Active transposons behave as “selfish” genetic elements. This characteristic is counteracted by the host's silencing machinery (blue arrow), which acts to methylate and silence transposon loci. This action can be blocked by miR820 (thin red line), which suppresses the host's silencing machinery and can drive host genome evolution (thick red arrow).