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. 2008 Oct;18(10):1602-9.
doi: 10.1101/gr.080127.108. Epub 2008 Jul 24.

Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening

Simon Moxon  1 , Runchun Jing, Gyorgy Szittya, Frank Schwach, Rachel L Rusholme Pilcher, Vincent Moulton, Tamas Dalmay
Affiliations

Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening

Simon Moxon et al. Genome Res. 2008 Oct.

Abstract

In plants there are several classes of 21-24-nt short RNAs that regulate gene expression. The most conserved class is the microRNAs (miRNAs), although some miRNAs are found only in specific species. We used high-throughput pyrosequencing to identify conserved and nonconserved miRNAs and other short RNAs in tomato fruit and leaf. Several conserved miRNAs showed tissue-specific expression, which, combined with target gene validation results, suggests that miRNAs may play a role in fleshy fruit development. We also identified four new nonconserved miRNAs. One of the validated targets of a novel miRNA is a member of the CTR family involved in fruit ripening. However, 62 predicted targets showing near perfect complementarity to potential new miRNAs did not validate experimentally. This suggests that target prediction of plant short RNAs could have a high false-positive rate and must therefore be validated experimentally. We also found short RNAs from a Solanaceae-specific foldback transposon, which showed a miRNA/miRNA*-like distribution, suggesting that this element may function as a miRNA gene progenitor. The other Solanaceae-specific class of short RNA was derived from an endogenous pararetrovirus sequence inserted into the tomato chromosomes. This study opens a new avenue in the field of fleshy fruit biology by raising the possibility that fruit development and ripening may be under miRNA regulation.

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Figures

Figure 1.
Figure 1.
Expression of conserved tomato miRNAs. Total RNA from different tissues was extracted, separated, and transferred to membranes. The membranes were hybridized to miRNA-specific probes or a U6-specific probe (shown on the right) to demonstrate equal loading. Membranes were stripped and reprobed; equal loading is shown once for each membrane. Numbers between brackets indicate the number of sequences found in the fruit (left) and leaf (right) libraries for each miRNA. Different size fruits were used for RNA extraction: F1, 1–3 mm; F2, 5–7 mm; F3, 7–11 mm; F4, 11–14 mm. Size markers on the left are 24- and 19-nt RNA oligonucleotides.
Figure 2.
Figure 2.
Target validation of conserved tomato miRNAs. 5′-RACE analysis was carried out for each predicted target gene. (Arrows) The 5′-ends of cleavage products. Cleavage sites outside of the displayed sequence are not shown. Target EST sequences are shown on top of the miRNA sequences.
Figure 3.
Figure 3.
Differentially expressed tomato short RNAs. Probes specific to potential miRNAs (tom72, NGM3, tom177, tom179, tom122, tom40, and tomtar3) or short RNAs that could not be mapped to the available genome sequence but cloned many times (top15, top12, top9, and top11) were hybridized to the same membranes shown on Figure 2. Sequences of short RNAs are provided in Supplemental Figure 2. The U6-specific probe shows equal loading. Different size fruits were used for RNA extraction: F1, 1–3 mm; F2, 5–7 mm; F3, 7–11 mm; F4, 11–14 mm. Size markers on the left are 24- and 19-nt RNA oligonucleotides.
Figure 4.
Figure 4.
Expression and target validation of new nonconserved tomato miRNAs. (A) Northern blot analysis of new miRNAs showed that sly-miR1918 and sly-miR1919 accumulate preferentially in the fruit. The U6 probe was used to show equal loading. Different size fruits were analyzed: F1, 1–3 mm; F2, 5–7 mm; F3, 7–11 mm; F4, 11–14 mm. (B) The result of target validation for three new miRNAs. (Arrows) The 5′ ends of cleavage products mapped inside the displayed sequence. Target EST sequences are shown on top of the miRNA sequences.
Figure 5.
Figure 5.
TAPIR-derived sRNAs. (A) Predicted secondary structure of one particular TAPIR element with lines representing the sRNA sequences mapping to the two arms of the hairpin. The color of the lines specifies the abundance of the sequences in the library. (B) Northern blot shows the accumulation of the two most abundant, overlapping sRNAs (exact positions are shown in Supplemental Fig. 6) from TAPIR elements. Membranes were stripped and reprobed for U6 to show equal loading. F1, 1–3 mm; F2, 5–7 mm; F3, 7–11 mm; F4, 11–14 mm.
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