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. 2012 Mar 19:3:46.
doi: 10.3389/fpls.2012.00046. eCollection 2012.

Non-Canonical Processing of Arabidopsis pri-miR319a/b/c Generates Additional microRNAs to Target One RAP2.12 mRNA Isoform

Lukasz Sobkowiak  1 , Wojciech Karlowski, Artur Jarmolowski, Zofia Szweykowska-Kulinska
Affiliations

Non-Canonical Processing of Arabidopsis pri-miR319a/b/c Generates Additional microRNAs to Target One RAP2.12 mRNA Isoform

Lukasz Sobkowiak et al. Front Plant Sci. .

Abstract

Arabidopsis miR319a/b/c primary transcripts are unusual due to the presence of a long stem and loop structure containing functional miR319a/b/c molecules. In our experiments carried out using high throughput sequencing (HTS), we have shown that additional microRNAs (miRNAs), miR319a.2/b.2/c.2 are generated from the upper part of the same hairpin structure. We have also found cognate miRNAa.2*/b.2*/c.2* to be present in the HTS results with a considerably lower number of reads. Northern hybridization revealed that miR319b.2 is mainly expressed in 35-day-old plant rosette leaves, as well as in stem and inflorescences of 42- and 53-day-old plants. Moreover, it carries multiple signatures of a functional miRNA, including as follows: (i) its biogenesis is HYL1-dependent; (ii) it is incorporated in a substantial amount into RISC complexes containing AGO1, AGO2, or AGO4 protein; (iii) 24 nt-long species of miR319b.2 have been found in inflorescences to be more abundant than 21 nt miR319b.2 species; (iv) it is present in various ratios to miR319b during plant development, which suggests the existence of a regulatory mechanism responsible for its biogenesis/processing; (v) there is an observed cross-species conservation of the miR319a/b/c stem nucleotide sequence extending beyond mature miRNA region; and (vi) all evidence suggests that intron-containing RAP2.12 mRNA isoform is the target for miR319b.2. All these features prompt us to claim miR319b.2 as a functional miRNA molecule.

Keywords: RAP2.12; gene expression; miRNA; pri-miRNA; splicing.

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Figures

Figure 1
Figure 1
Stem and loop structures of Arabidopsis pre-miR319 precursors. Sequences of miR319a, b, and c are in green, sequences representing miRNA319a.2, b.2, and c.2 are in red, and star sequences are in black circles, respectively.
Figure 2
Figure 2
Quantitative real-time PCR profiling of pri-miRNAs from MIR319 family in different developmental stages in wild type A. thaliana plants (14-, 20-, 35-, 42-, and 53-day-old plants). (A–C) Expression patterns of pri-miR319a, pri-miR319b, and pri-miR319c, respectively. *Not detected, ^log10 calculated from the fold change of particular pri-miRNAs standardized to the PP2A transcript level (At1g69960). Because the expression of pri-miR319a/b/c is in most cases lower than the level of PP2A transcript, the graph was rescaled according to the following formula: abs(xmax-x), where abs denotes absolute value, xmax represents the lowest integer value of relative expression level in the original graph and x represents actual expression level for a given pri-miRNA in the particular developmental stage. Real-time PCR for all pri-miR319 was carried out in three biological replicates. Thin black lines represent SD.
Figure 3
Figure 3
The phenotypes of 42-day-old A. thaliana wild type, ΔmiR319b (Salk_037093), and miR319boe (Salk_059451) mutant plant (A). RT-PCR profiling of the pri-miR319b level. (B) The upper panel shows the increased level of primary transcript in the miR319boe mutant and the lower panel – the lack of transcript in the case of ΔmiR319b mutant plants. Northern blot hybridization (C,D) of miR319 and miR319b.2 in the wild type, miR319boe and ΔmiR319b mutant plants. RNA was isolated from stems [left panel (C)] and inflorescenses [right panel (D)]. For better visualization of miR319b.2 hybridization signals, these blots were longer exposed than in the case of miR319b.
Figure 4
Figure 4
Detection of the mature miR319 and miR319b.2 in the wild type A. thaliana plants. Northern hybridization was performed with total RNA enriched for small RNAs from 14-day-old seedlings (A), 20-day-old rosette leaves (B), 35-day-old plants (roots, rosette leaves, and stems) (C), 42-day-old (D), and 53-day-old plants (E) (roots, rosette leaves, stems, and inflorescences) using the probe for miR319a/b and miR319b.2. Biogenesis of miR319b.2 is HYL1-dependent (F). In (F), hybridization was carried out using RNA isolated from the stems of 42-day-old plants. The ratio between 319b and 319b.2 signals is indicated in (C–E), and between miR319b or miR319b.2 in wild type and hyl1-2 mutant plants in (F). For better visualization of miR319b.2 hybridization signals, these blots were longer exposed than in the case of miR319b. The probe for U6 snRNA was used as RNA loading control. (G) Shows nucleotide sequences of miR319a/b/c, and miR319a.2/b.2/c.2, respectively.
Figure 5
Figure 5
The structure of RAP2.12 gene (A) and its two mRNA isoforms (B). The predicted slicing site within intron-retaining mRNA isoform is marked and enlarged. Black arrows point to predicted slicing site and identified products of 5′-RACE carried out for RAP2.12 slicing products. (C) The agarose gel showing the 3′-RAP2.12 intron-containing mRNA isoform cleavage products in wt plants, miR319boe, and ΔmiR319b mutant plants. Arrow points to the expected length of 5′-RACE product, star depicts degradation products obtained during 5′-RACE. In each line, five 5′-RACE reaction products were pooled together and loaded on gel.
Figure 6
Figure 6
Real-time PCR analyses performed for two RAP2.12 mRNA isoforms in 42-day-old plants. Fully spliced isoform (black bars) was amplified using primers flanking exon–exon junction and the intron-retaining isoform (gray bars) was amplified with primers flanking predicted slicing site (A, B). Relative expression level of both mRNA isoforms of wild type and mutant hyl1-2, se-1, miR319boe, and ΔmiR319b plants, were analyzed in stems and inflorescences, respectively. Ct values for all mRNA transcripts were normalized against the PP2A (At1g69960) Ct value.
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