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Identification of MIR390a precursor processing defective mutants in Arabidopsis by direct genome sequencing

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https://ir.library.oregonstate.edu/concern/articles/zp38wd185

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  • Transacting siRNA (tasiRNA) biogenesis in Arabidopsis is initiated by microRNA (miRNA) –guided cleavage of primary transcripts. In the case of TAS3 tasiRNA formation, ARGONAUTE7 (AGO7)– miR390 complexes interact with primary transcripts at two sites, resulting in recruitment of RNA-DEPENDENT RNA POLYMERASE6 for dsRNA biosynthesis. An extensive screen for Arabidopsis mutants with specific defects in TAS3 tasiRNA biogenesis or function was done. This yielded numerous ago7 mutants, one dcl4 mutant, and two mutants that accumulated low levels of miR390. A direct genome sequencing-based approach to both map and rapidly identify one of the latter mutant alleles was developed. This revealed a G-to-A point mutation (mir390a-1) that was calculated to stabilize a relatively nonpaired region near the base of the MIR390a foldback, resulting in misprocessing of the miR390/ miR390* duplex and subsequent reduced TAS3 tasiRNA levels. Directed substitutions, as well as analysis of variation at paralogous miR390-generating loci (MIR390a and MIR390b), indicated that base pair properties and nucleotide identity within a region 4–6 bases below the miR390/miR390* duplex region contributed to the efficiency and accuracy of precursor processing.
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  • Cuperus, J. T., Montgomery, T. A., Fahlgren, N., Burke, R. T., Townsend, T., Sullivan, C. M., Carrington, J. C. (2010). Identification of MIR390a precursor processing defective mutants in Arabidopsis by direct genome sequencing. Proceeding of the National Acedemy of Science, 107(1), 466-471
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  • 107
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  • 1
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  • We thank Sarah Dvorak for assistance with the genetic screen and technical assistance. We also thank Detlef Weigel, Korbinian Schneeberger, and Richard Clark for helpful discussion, genomic sequencing test data sets, and SNP data; and we thank Nina Fedoroff, Mike Axtell, Detlef Weigel, and Javier Palatnik for sharing data and manuscripts prior to publication.This work was supported by grants from the National Science Foundation (MCB-0618433), National Institutes of Health (AI43288) and United States Department of Agriculture–National Research Initiative (2006-35301-17420)
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