A diverse set of microRNAs and microRNA-like small RNAs in developing rice grains

Abstract

Endogenous small RNAs, including microRNAs (miRNAs) and short-interfering RNAs (siRNAs), function as post-transcriptional or transcriptional regulators in plants. miRNA function is essential for normal plant development and therefore is likely to be important in the growth of the rice grain. To investigate the roles of miRNAs in rice grain development, we carried out deep sequencing of the small RNA populations of rice grains at two developmental stages. In a data set of approximately 5.5 million sequences, we found representatives of all 20 conserved plant miRNA families. We used an approach based on the presence of miRNA and miRNA* sequences to identify 39 novel, nonconserved rice miRNA families expressed in grains. Cleavage of predicted target mRNAs was confirmed for a number of the new miRNAs. We identified a putative mirtron, indicating that plants may also use spliced introns as a source of miRNAs. We also identified a miRNA-like long hairpin that generates phased 21 nt small RNAs, strongly expressed in developing grains, and show that these small RNAs act in trans to cleave target mRNAs. Comparison of the population of miRNAs and miRNA-like siRNAs in grains to those in other parts of the rice plant reveals that many are expressed in an organ-specific manner.

Figure 1.

(A) Ratio (6–10 DAF/1-5 DAF) of the normalized miRNA sequence reads of the known miRNA families with at least 10 reads in total in the Illumina data set. The raw sequence reads of each miRNA family is shown on top of the bars with numerator and denominator representing number of reads in 6–10 DAF and 1–5 DAF grains, respectively. (B) Mapping of miRNA-guided cleavage sites in predicted target genes. Predicted cleavage sites are indicated by a bold italic nucleotide at position 10 relative to the 5′ end of the miRNA. Mapped cleavage positions are indicated by arrows with the frequency amongst 5′ RACE clones sequenced. (C) Northern blot detection of expression of miRNAs in different tissues. Uniform RNA loading was demonstrated by reprobing blots with 5S rRNA (representative blot shown). (S) Shoots; (R) roots; (G1) 1–5 DAF grains; (G6) 6–10 DAF grains.

Figure 2.

miRNA-like long hairpins producing phased 21-nt small RNAs. (A) Hairpin of the pre-miR436. In total, 13,141 and 2439 sequence reads were generated from the 5′ and the 3′ arms of the hairpin, respectively, and only 19 reads were mapped to the antisense strand. Of these sequences, 91.2% were 21 nt in length and 95.9% of these 21-nt small RNAs fell in phases of 21-nt spacing. The number of reads for all small RNAs with a common 5′ terminus (regardless of size) is plotted, with red and blue bars representing sequences from the 5′ and the 3′ arms, respectively. The 21-nt phases corresponding to the 5′ arm are indicated by the vertical dotted lines. Phase identifiers are shown above and below the hairpin. Nucleotide positions shown on the X-axis are based on the 5′ arm of the hairpin starting from the 5′ end of phase P12_5′. miR436 and miR436* are shown in green and orange, respectively. (B) A miRNA-like long hairpin generated from the antisense strand of LOC_Os06g21900. In total, 59,150 sequence reads were generated (∼60% from the 5′ arm and ∼40% from the 3′ arm; ∼97% of the reads were from 6–10 DAF grains), including 97 reads mapped to the antisense strand; 54,857 (92.7%) reads were 21 nt in length and 93.9% of these 21-nt small RNAs were in exact 21-nt phases. The frequency and distribution of small RNAs is plotted as described in A. (C) 5′ RACE validation of cleavage of predicted target mRNAs by P6_5′ and P6_3′. (D) Expression of small RNAs from the newly identified long hairpin in different tissues detected by a probe antisense to the 3′ arm of the hairpin (218 nt in length). The blot used was the same as that for miR1861 shown in Figure 1C. (S) Shoots; (R) roots; (G1) 1–5 DAF grains; (G6) 6–10 DAF grains.

Figure 3.

miR1429.2 is a putative mirtron. (A) Pre-miR1429.2 (shown in capital letters; adjacent exon sequences are shown in lowercase letters; miR1429 is underlined) with aligned small RNAs. The mature miRNA (miR1429.2 in red) and its miRNA* (in blue), and the termini of the intron (vertical pink lines), are indicated with the predicted secondary structure shown in bracket notation. Positions of the annotated splicing donor and acceptor were supported by cDNA sequence AB101650 (sequences flanking the intron are shown in italic at the first row). The 3′ terminal nucleotide in the last two small RNAs, which do not match the genomic sequence, might be generated by untemplated addition. (B) Predicted secondary structure of pre-miR1429.2 showing 2-nt 3′ overhangs.