Multiple distinct small RNAs originate from the same microRNA precursors

Abstract

Background: MicroRNAs (miRNAs), which originate from precursor transcripts with stem-loop structures, are essential gene expression regulators in eukaryotes.

Results: We report 19 miRNA precursors in Arabidopsis that can yield multiple distinct miRNA-like RNAs in addition to miRNAs and miRNA*s. These miRNA precursor-derived miRNA-like RNAs are often arranged in phase and form duplexes with an approximately two-nucleotide 3'-end overhang. Their production depends on the same biogenesis pathway as their sibling miRNAs and does not require RNA-dependent RNA polymerases or RNA polymerase IV. These miRNA-like RNAs are methylated, and many of them are associated with Argonaute proteins. Some of the miRNA-like RNAs are differentially expressed in response to bacterial challenges, and some are more abundant than the cognate miRNAs. Computational and expression analyses demonstrate that some of these miRNA-like RNAs are potentially functional and they target protein-coding genes for silencing. The function of some of these miRNA-like RNAs was further supported by their target cleavage products from the published small RNA degradome data. Our systematic examination of public small-RNA deep sequencing data from four additional plant species (Oryza sativa, Physcomitrella patens, Medicago truncatula and Populus trichocarpa) and four animals (Homo sapiens, Mus musculus, Caenorhabditis elegans and Drosophila) shows that such miRNA-like RNAs exist broadly in eukaryotes.

Conclusions: We demonstrate that multiple miRNAs could derive from miRNA precursors by sequential processing of Dicer or Dicer-like proteins. Our results suggest that the pool of miRNAs is larger than was previously recognized, and miRNA-mediated gene regulation may be broader and more complex than previously thought.

Figure 1

Four miRNA precursors that can generate multiple miRNA-like RNAs. (a-d). Three miRNA precursors with miRNA-like RNAs in the upper arms close to the loops of their hairpins (a, c, d), and a miRNA precursor with miRNA-like RNAs in the lower arm of its hairpin (b). Note that miRNA-miRNA* duplexes for miRNA-like RNAs, with approximately two-nucleotide 3'-end overhangs, appear on the miR159, miR169m and miR319b precursors. The previously annotated miRNAs were named as miRn.1 (see main text for detail) and those miRNA-like RNAs having less than four reads were not named. For clarity, miR169m.2* and miR319b.2* are also indicated though the numbers of reads mapped to them were below the cutoff threshold of 5.

Figure 2

miRNA-like RNAs from the miR447a precursor. (a) The precursor fold-back structure and sequencing reads mapped to miR447a.1 (that is, miR447a) and the individual miRNA-like RNAs. For clarity, miR447a.3* and miR447a.1* are indicated though the numbers of reads mapped to them were below the cutoff threshold of 5. (b) Distribution of sequencing reads along the precursor. (c) Expression of miR447a.1 and miR447a.3 in various Arabidopsis mutants of small RNA pathways. (d) Expression of miR447a.3, miR447a.1, and miR447a.2-3p under the challenge of three Pst strains (hrcC, avrRpt2 and EV) and in a mock control at 6 and 14 hpi.

Figure 3

miRNA-like RNAs from the miR822 precursor. (a) The precursor fold-back structure and sequencing reads mapped to miR822.1 (that is, miR822) and individual miRNA-like RNAs. (b) Distribution of sequencing reads along the precursor. For clarity, miR822.5* is indicated though the number of reads mapped to it is below the cutoff threshold of 5. (c) Expression of miR822.1 and miR822.2 in various Arabidopsis mutants of small RNA pathways. (d) Expression of miR822.2, miR822.1, miR822.3, miR822.1* and miR822.3* under the challenge of three Pst strains (hrcC, avrRpt2 and EV) and in a mock control at 6 and 14 hpi.

Figure 4

miRNA-like RNAs from the miR839 precursor. (a) The precursor fold-back structure and sequencing reads corresponding to the individual miRNA-like RNAs miR839.1 (that is, miR839) and miR839.1* (that is, miR839*). Note that miR839.1*, miR839.2 and miR839.3 have more sequencing reads than miR839.1. For clarity, miR839.3* was is though the number of reads mapped to it is below the cutoff threshold of 5. (b) Distribution of sequencing reads along the precursor. (c) Expression of miR839.1, miR839.2 and miR839.3 under the challenge of three Pst strains (hrcC, avrRpt2 and EV) and in a mock control at 6 and 14 hpi.

Figure 5

Accumulation of miR447a.3 and miR822.2 in a mutant of the small-RNA methyltransferase gene HEN1. WT, wild type. U6, the control, shows sRNA equal loading.

Figure 6

Negative correlation between the expression of selected miRNA-like RNAs and their targets. (a) The expression of targets of miR169i.2-3p, miR169j.2, miR447a.3, miR839.2 and miR839.3 in a dcl1-9 mutant relative to that in the Ler wild type (WT), measured by realtime RT-PCR. (b) The expression level of the miR822.4-5p target in a dcl4-2 mutant relative to the Col-0 wide type. (c) The expression of two miR447a.3 targets and one miR839.2 target under the challenge of Pst DC3000 (avrRpt2) relative to that in the mock treatment. Actin was used as an internal control for delta Ct calculation. Error bars correspond to standard deviation data from three independent reactions. The experiments were repeated on three sets of biological samples and similar results were obtained. (d) Alignments of selected miRNA-like RNAs and some of their mRNA targets whose expression was compared in Dicer mutants and in the wide type (a, b) and under bacterial infection and under mock infection (c). Included are alignment scores and P-values of target signatures if miRNA-like RNAs had target degradation products in the three small RNA degradome datasets. The arrows are the target cleavage sites detected in the degradome data.

Figure 7

Conservation of miRNA-like RNAs and miRNAs. The newly identified miRNA-like RNAs are in red and the annotated miRNAs and miRNA*s are in blue. Also included are the consensus sequences. (a) Conservation of miR159a.2-5p/miR159a.2-3p and miR159a.1/miR159a.1* (that is, miR159a/miR159a*) in four plants, A. thaliana (ath), O. sativa (osa), P. patens (ppt) and M. truncatula (mtr). (b) Conservation of miR319.2/miR319b.2* and miR319.1/miR319b.1* (that is, miR319/miR319b*) in four plants, Arabidopsis, rice, Medicago and P. trichocarpa (ptc). (c) Conservation of miR7-1 in human and miR7a-1 in mouse.