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. 2013 Jul;14(7):622-8.
doi: 10.1038/embor.2013.62. Epub 2013 May 17.

Introns of plant pri-miRNAs enhance miRNA biogenesis

Dawid Bielewicz  1 , Malgorzata Kalak, Maria Kalyna, David Windels, Andrea Barta, Franck Vazquez, Zofia Szweykowska-Kulinska, Artur Jarmolowski
Affiliations

Introns of plant pri-miRNAs enhance miRNA biogenesis

Dawid Bielewicz et al. EMBO Rep. 2013 Jul.

Abstract

Plant MIR genes are independent transcription units that encode long primary miRNA precursors, which usually contain introns. For two miRNA genes, MIR163 and MIR161, we show that introns are crucial for the accumulation of proper levels of mature miRNA. Removal of the intron in both cases led to a drop-off in the level of mature miRNAs. We demonstrate that the stimulating effects of the intron mostly reside in the 5'ss rather than on a genuine splicing event. Our findings are biologically significant as the presence of functional splice sites in the MIR163 gene appears mandatory for pathogen-triggered accumulation of miR163 and proper regulation of at least one of its targets.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
The intron of MIR163 stimulates the biogenesis of miR163. (A) Schematic representation of MIR163 gene variants used. The splicing sites and position of proximal and distal poly(A) sites are shown. (B) Level of pri-miR163 (upper panel) and miR163 (lower panel) recorded in different organs of the mir163-2 mutant. U6 snRNA serves as a loading control. (C) Level of pri-miR163 (upper panel), pri-miR163 splicing variants (middle panel) and miR163 (lower panel) recorded in lines expressing MIR163 variants shown in B. IVSwt.1 and IVSwt.2 are independent lines with intron-containing MIR163; IVS.1 and ΔIVS.2 are independent lines with intron-less MIR163. U6 snRNA serves as a loading control. Error bars indicate s.d. (n=3), and asterisks indicate a significant difference between the indicated sample and control wild type plants (Mann–Whitney test, P<0.05). NTC, non-template control.
Figure 2
Figure 2
Mutations of splice sites in MIR163 affects the biogenesis of miR163. (A) Schematic representation of MIR163 gene variants used. The splicing sites and position of proximal and distal poly(A) sites are shown. The sequences of wt and mutated splice sites are also shown. (B) Level of pri-miR163 splice variants (upper panel) and miR163 (lower panel) recorded in lines expressing MIR163 gene variants. U6 snRNA serves as a loading control. (C) Ratio of proximal versus distal poly(A) site usage determined in wt plants and MIR163 variant lines. IVSΔ5′ss.1 and IVSΔ5′ss.2 are independent lines in which the MIR163 5′ss was mutated; IVSΔ3′ss.1 and IVSΔ3′ss.2 are independent lines in which the MIR163 3′ss was mutated; IVSmut.1 and IVSmut.2 are independent in which the MIR163 5′ss and 3′ss are mutated. Error bars indicate s.d. (n=3), and asterisks indicate a significant difference between the indicated sample and control wt plants (Mann–Whitney test, P<0.05). wt, wild type.
Figure 3
Figure 3
SR proteins affect biogenesis of miR163. Efficiency of pri-miR163 splicing recorded by semiquantitative RT-PCR (upper panel) and by final-point RT-PCR (middle panel) in sr mutants (rs31-1, rs2z33-1, sr34-1 and scl30a-1) and miRNA biogenesis mutants (hyl1-2, se-1, cbp20, cbp80 and cbc, which is the cbp20/cbp80 double mutant). The level of miR163 is given in the lower panel. U6 snRNA serves as a loading control. Error bars indicate s.d. (n=3), and asterisks indicate a significant difference between the indicated sample and control wt plants (Mann–Whitney test, P<0.05). NTC, non-template control.
Figure 4
Figure 4
The accumulation of miR163 after pathogen infection depends on the presence of functional splice sites in the MIR163 gene. (A) Identification of miR163-guided cleavage sites in At1g66690 mRNAs. The cleavage site was detected in wt and in the xrn4-3 mutant. The PCR products at the expected size are marked with arrows. The upper sequence shows the exact cleavage site identified and the sequence of the primer used for 5′RACE. The number of clones (8/8) sequenced are given. (B) Level of the At1g66690 mRNA recorded in wt and in the miRNA biogenesis mutants: hyl1-2, se-1, cbp20, cbp80 and cbc (that is, cbp20/cbp80 double mutant). Error bars indicate s.d. (n=3), and asterisks indicate a significant difference between the indicated sample and control wt plants (Mann–Whitney test, P<0.05). (C) Level of pri-miR163 (upper panel), of miR163 (middle panel), and of its validated mRNA target (lower panel) recorded in wt and IVSmut plants after 0, 24 or 72 h infection with P. syringae DC3000. Error bars indicate s.d. (n=3). One asterisk indicates a significant difference between the indicated sample and wt plants at the 0 h time point, and double asterisk indicates a significant difference between the indicated sample and IVSmut plants at the 0 h time point (Mann–Whitney test, P<0.05). wt, wild type.
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