期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 107, 期 1, 页码 466-471出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0913203107
关键词
high-throughput sequencing; miRNA; trans-acting siRNA
资金
- National Science Foundation [MCB-0618433]
- National Institutes of Health [AI43288]
- United States Department of Agriculture-National Research Initiative [2006-35301-17420]
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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