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MIR846 and MIR842 comprise a cistronic MIRNA pair that is regulated by abscisic acid by alternative splicing in roots of Arabidopsis

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An Erratum to this article was published on 19 April 2013

Abstract

MicroRNAs (miRNAs) are ~21-nucleotide long endogenous small RNAs that regulate gene expression through post-transcriptional or transcriptional gene silencing and/or translational inhibition. miRNAs can arise from the “exon” of a MIRNA gene, from an intron (e.g. mirtrons in animals), or from the antisense strand of a protein coding gene (natural antisense microRNAs, nat-miRNAs). Here we demonstrate that two functionally related miRNAs, miR842 and miR846, arise from the same transcription unit but from alternate splicing isoforms. miR846 is expressed only from Isoform1 while in Isoforms2 and -3, a part of pre-miR846 containing the miRNA* sequence is included in the intron. The splicing of the intron truncates the pre-MIRNA and disrupts the expression of the mature miR846. We name this novel phenomenon splicing-regulated miRNA. Abscisic acid (ABA) is shown to mediate the alternative splicing event by reducing the functional Isoform1 and increasing the non-functional Isoform3, thus repressing the expression of miR846 concomitant with accumulation of an ABA-inducible target jacalin At5g28520 mRNA, whose cleavage was shown by modified 5′-RACE. This regulation shows the functional importance of splicing-regulated miRNA and suggests possible mechanisms for altered ABA response phenotypes of miRNA biogenesis mutants. Arabidopsis lyrata-MIR842 and Aly-MIR846 have conserved genomic arrangements with A. thaliana and candidate target jacalins, similar primary transcript structures and intron processing, and better miRNA–miRNA* pairings, suggesting that the interactions between ABA, MIR842, MIR846 and jacalins are similar in A. lyrata. Together, splicing-regulated miRNAs, nat-miRNAs/inc-miRNAs and mirtrons illustrate the complexity of MIRNA genes, the importance of introns in the biogenesis and regulation of miRNAs, and raise questions about the processes and molecular mechanisms that drive MIRNA evolution.

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Acknowledgments

The authors thank Qingjun Luo, Amandeep Mittal and Yingwen Jiang for helpful discussions, and the TTU High Performance Computing Center for use of the Hrothgar cluster. This work was supported by the National Institutes of Health grant GM077245 to C.D.R, and a Texas Tech University AT&T Chancellor’s Fellowship to F.J. The funders had no role in the study design, in the collection, analysis, or interpretation of data, or in the writing of the manuscript or decision to submit the manuscript for publication.

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  1. Department of Biological Sciences, Texas Tech University, Lubbock, TX, 79409-3131, USA

    Fan Jia & Christopher D. Rock

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Correspondence to Christopher D. Rock.

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11103_2013_15_MOESM1_ESM.pptx

GUS staining of wild type (A, C, E) and Pro-MIR842_MIR846:GUS transgenic plants (B, D, F) showing similar background staining in inflorescence (A, B), the junction between siliques and petioles (C, D), and in the midrib of rosette leaves (E, F). (PPTX 28184 kb)

11103_2013_15_MOESM2_ESM.pptx

Arabidopsis virtual expression analysis from microarray transcriptome eFP data (http://bar.utoronto.ca/) for AT5G28520 visualizing root-specific induction for seedlings treated with 300 mM Mannitol, with 150 mM NaCl (A), or treated with ABA in (B). (PPTX 922 kb)

11103_2013_15_MOESM3_ESM.pptx

Alignment between A. thaliana and A. lyrata genomic sequences containing MIR842 and MIR846. Gray lines represent pre-miRNAs. Black boxes represent mature miRNA or miRNA* sequences. Black arrowheads represent GT..AG intron 5’ and 3’ splice boundaries. Alignment performed with tcoffee (www.tcoffee.org). (PPTX 1310 kb)

11103_2013_15_MOESM4_ESM.pptx

A.lyrata jacalin homologs carrying miR846 complementary sites. Numbers above upper sequences represent nt positions in the mRNA, according to Genbank ID annotations. Vertical lines represent perfect matches. “o” represents a G:U wobble. X represents a mismatch. (PPTX 5758 kb)

11103_2013_15_MOESM5_ESM.pptx

Models of (A) a splicing regulated miRNA, (B) a Nat-miRNA/Inc-miRNA, and (C) a mirtron. Thick black lines represent exons; thick gray lines represent introns; thin gray lines represent flanking sequences; stem-loops represent pre-miRNAs; horizontal lines, Watson–Crick base pairs. (PPTX 907 kb)

11103_2013_15_MOESM6_ESM.pptx

A miR846 variant can target AT5G28520. (A) A part of miR846 stem-loop sequence. The mature miR846 sequence is marked in pink. (B) Base pairing between the potential miR846 variant and AT5G28520 showing extended complementarity at the 5′ end. The mature miR846 sequence is marked in pink. Red underline indicates the variant. Numbers represent positions in the mRNA. Vertical lines represent perfect matches. “o” represents a G:U wobble. X represents a mismatch. The thick black line represents the cleavage site shown by modified 5′-RACE. (PPTX 204 kb)

11103_2013_15_MOESM7_ESM.xlsx

Potential targets of miR842 or miR846 identified with “psRNATarget” (http://plantgrn.noble.org/psRNATarget/) (Dai and Zhao, 2011). (XLSX 20 kb)

Oligonucleotide primers used in this study. (XLSX 10 kb)

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Jia, F., Rock, C.D. MIR846 and MIR842 comprise a cistronic MIRNA pair that is regulated by abscisic acid by alternative splicing in roots of Arabidopsis. Plant Mol Biol 81, 447–460 (2013). https://doi.org/10.1007/s11103-013-0015-6

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