Gene structures and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs

Abstract

Arabidopsis thaliana HYL1 is a nuclear double-stranded RNA-binding protein involved in the maturation of pri-miRNAs. A quantitative real-time PCR platform for parallel quantification of 176 pri-miRNAs was used to reveal strong accumulation of 57 miRNA precursors in the hyl1 mutant that completely lacks HYL1 protein. This approach enabled us for the first time to pinpoint particular members of MIRNA family genes that require HYL1 activity for efficient maturation of their precursors. Moreover, the accumulation of miRNA precursors in the hyl1 mutant gave us the opportunity to carry out 3' and 5' RACE experiments which revealed that some of these precursors are of unexpected length. The alignment of HYL1-dependent miRNA precursors to A. thaliana genomic sequences indicated the presence of introns in 12 out of 20 genes studied. Some of the characterized intron-containing pri-miRNAs undergo alternative splicing such as exon skipping or usage of alternative 5' splice sites suggesting that this process plays a role in the regulation of miRNA biogenesis. In the hyl1 mutant intron-containing pri-miRNAs accumulate alongside spliced pri-miRNAs suggesting the recruitment of HYL1 into the miRNA precursor maturation pathway before their splicing occurs.

Figure 1.

Detection of some of the mature miRNAs, which biogenesis strongly depends on HYL1 protein action. Northern hybridization was performed with total RNA from 35-day-old rosette leaves of wild-type plants (WT) and hyl1 mutant (hyl1). The probe for U6 snRNA was used as RNA-loading control.

Figure 2.

Agarose gel electrophoresis of full-length miRNA transcripts of MIRNA genes. (A) Intronless MIRNA genes 160b and 169f. (B) MIRNA genes containing one intron: 158a and 166a. RT-PCR products, obtained using primers designed on the bases of distal sequences of the longest 5′ and 3′ RACE products are shown. WT and hyl1, cDNA templates obtained from wild-type and hyl1 plants, respectively, with concentration standardized by real-time PCR to POLYUBIQUITIN 10 (UBQ10, At4g05320) expression level; control (−) refers to RT-PCR reaction without template; control (+), PCR reaction carried out with A. thaliana wild-type genomic DNA as a positive control. M, GeneRuler 100-bp DNA Ladder.

Figure 3.

Diagram showing spectra of miRNA precursors. (A) miRNA precursors within the miR160 family. (B) miRNA precursors including forms resulting from alternative splicing events: exon skipping (pri-miR156c) and alternatively chosen 5′ splice site (pri-miR166b). Black barrel and black barrel with a star depict mature miRNA and miRNA* sequence, respectively.

Figure 4.

The percentage (%) of miRNA precursor splicing forms in wild-type plants (WT) and hyl1 mutant (hyl1). (A) miR158a and (B) miR161. The comparison of the relative amounts of intron-containing (indicated dark gray) and intronless (light gray) pri-miRNAs shows the predominant participation of intron-containing forms both in wild-type and hyl1 mutant plants. Data are the mean ± standard deviation of median values of each biological replicates. The experiments were performed in two or three biological replicates (with two to five technical replicates for each).

Figure 5.

Analysis of pri-miRNA and pre-mRNA splicing in wild-type plants, the mutants hyl1, cbp20, abh1/cbp80, and the double mutant cbp20xabh1/cbp80. (A–B) Both unspliced and spliced forms of pri-miRNAs accumulate in hyl1 mutant in comparison to wild-type plant. The asterisk in B marks an additional unidentified DNA band. (C–E) There is no significant difference in the accumulation of intron-containing mRNA precursors in wild-type and hyl1 mutant background. (F) The amount of cDNA was standardized by RT-PCR to the ACTIN2 (ACT2, At3g18780) expression level. gDNA refers to the PCR reaction carried out with A. thaliana wild-type genomic DNA as a positive control. The graphic presentations on the right indicate the respective transcripts with their exon (gray box) and intron (line) organizations.