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A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants
Published on: January 21, 2020
The Arabidopsis MOS4-Associated Complex Promotes MicroRNA Biogenesis and Precursor Messenger RNA Splicing
Tianran Jia1, Bailong Zhang1, Chenjiang You1,2
1Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, University of California, Riverside, California 92521.
Abstract:
In Arabidopsis thaliana, the MOS4-ASSOCIATED COMPLEX (MAC) is required for defense and development. The evolutionarily conserved, putative RNA helicase MAC7 is a component of the Arabidopsis MAC, and the human MAC7 homolog, Aquarius, is implicated in pre-mRNA splicing. Here, we show that mac7-1, a partial loss-of-function mutant in MAC7, and two other MAC subunit mutants, mac3a mac3b and prl1 prl2 (pleiotropic regulatory locus), exhibit reduced microRNA (miRNA) levels, indicating that MAC promotes miRNA biogenesis. The mac7-1 mutant shows reduced primary miRNA (pri-miRNA) levels without affecting miRNA gene (MIR) promoter activity or the half-life of pri-miRNA transcripts. As a nuclear protein, MAC7 is not concentrated in dicing bodies, but it affects the localization of HYPONASTIC LEAVES1 (HYL1), a key protein in pri-miRNA processing, to dicing bodies. Immunoprecipitation of HYL1 retrieved 11 known MAC subunits, including MAC7, indicating association between HYL1 and MAC. We propose that MAC7 links MIR transcription to pri-miRNA processing. RNA-seq analysis showed that downregulated genes in MAC subunit mutants are mostly involved in plant defense and stimulus responses, confirming a role of MAC in biotic and abiotic stress responses. We also discovered global intron retention defects in mutants in three subunits of MAC, thus linking MAC function to splicing in Arabidopsis.
Insights
The MOS4-associated complex (MAC) in Arabidopsis promotes microRNA (miRNA) biogenesis and is crucial for plant defense and development. MAC subunit mutants show reduced miRNA levels and global intron retention, linking MAC to splicing and stress responses.
Area of Science:
- Molecular Biology
- Plant Science
- Genetics
Background:
- The MOS4-associated complex (MAC) is essential for defense and development in Arabidopsis thaliana.
- MAC7, a component of Arabidopsis MAC, is an evolutionarily conserved RNA helicase, with its human homolog Aquarius linked to pre-mRNA splicing.
- MicroRNAs (miRNAs) are key regulators of gene expression in plants.
Purpose of the Study:
- To investigate the role of MAC7 and other MAC subunits in miRNA biogenesis and plant stress responses.
- To elucidate the mechanism by which MAC affects miRNA processing.
- To determine the connection between MAC function and pre-mRNA splicing in Arabidopsis.
Main Methods:
- Analysis of loss-of-function mutants (mac7-1, mac3a mac3b, prl1 prl2) for miRNA levels.
- Measurement of primary miRNA (pri-miRNA) levels and MIR gene promoter activity.
- Subcellular localization studies of MAC7 and HYL1.
- Immunoprecipitation assays to identify MAC-HYL1 interactions.
- RNA-sequencing (RNA-seq) to analyze gene expression and intron retention.
Main Results:
- Mutants in MAC subunits (mac7-1, mac3a mac3b, prl1 prl2) exhibit significantly reduced miRNA levels.
- MAC7 affects the localization of HYPONASTIC LEAVES1 (HYL1) to dicing bodies, a key step in pri-miRNA processing.
- Immunoprecipitation confirmed the association of MAC subunits, including MAC7, with HYL1.
- RNA-seq revealed that downregulated genes in MAC mutants are primarily involved in plant defense and stimulus responses.
- Global intron retention defects were observed in mutants of three MAC subunits.
Conclusions:
- The MAC complex, particularly MAC7, plays a vital role in promoting miRNA biogenesis by facilitating pri-miRNA processing through interaction with HYL1.
- MAC is essential for plant defense and responses to biotic and abiotic stimuli.
- The MAC complex is involved in regulating pre-mRNA splicing, as evidenced by global intron retention defects in MAC subunit mutants.
- MAC acts as a molecular link connecting miRNA transcription to pri-miRNA processing and also plays a role in splicing.
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