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Differentiated Truncation and Uridylation Patterns of miRNAs in Arabidopsis and Rice hen1 Mutants Suggest miRNAs May Re-program RISC

ABSTRACT: miRNA levels depend on both biogenesis and turnover. The methyltransferase HEN1 stabilizes plant miRNAs, animal piRNAs, and siRNAs in both kingdoms via 3' terminal methylation. Loss of HEN1 in plants results in non-templated oligo-uridylation and accelerated degradation of miRNAs. In hen1 mutants from Arabidopsis and rice, we found that the patterns of miRNA truncation and uridylation differ substantially among miRNA families, but such patterns for the same miRNA are conserved between species. miR166 and miR163 are truncated predominantly to ~17 and ~16 nt, and subsequently recover via uridylation to approximately their original sizes, 21 and 24 nt, suggesting that in these cases miRNA truncation triggers uridylation. miR171 is untruncated but uridylated to 22 nt in hen1 mutants, gaining the ability to trigger production of phased, secondary siRNAs. Truncated and tailed variants were bound by ARGONAUTE1 (AGO1) in hen1, implying that these events occur while miRNAs are still bound by AGO1. Unexpectedly, a portion of miR158 in wildtype remains unmethylated and thus subject to uridylation and destabilization, suggesting that plants naturally utilize miRNA methylation to modulate miRNA accumulation. Our results suggest that the AGO1-containing RISC complex may undergo programming to reflect each bound miRNA, determining a defined, distinct decay destiny.

ORGANISM(S): Arabidopsis thaliana Oryza sativa Zea mays

PROVIDER: GSE35562 | GEO | 2013/07/22

SECONDARY ACCESSION(S): PRJNA152685

REPOSITORIES: GEO

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Project description:miRNA levels depend on both biogenesis and turnover. The methyltransferase HEN1 stabilizes plant miRNAs, animal piRNAs, and siRNAs in both kingdoms via 3' terminal methylation. Loss of HEN1 in plants results in non-templated oligo-uridylation and accelerated degradation of miRNAs. In hen1 mutants from Arabidopsis and rice, we found that the patterns of miRNA truncation and uridylation differ substantially among miRNA families, but such patterns for the same miRNA are conserved between species. miR166 and miR163 are truncated predominantly to ~17 and ~16 nt, and subsequently recover via uridylation to approximately their original sizes, 21 and 24 nt, suggesting that in these cases miRNA truncation triggers uridylation. miR171 is untruncated but uridylated to 22 nt in hen1 mutants, gaining the ability to trigger production of phased, secondary siRNAs. Truncated and tailed variants were bound by ARGONAUTE1 (AGO1) in hen1, implying that these events occur while miRNAs are still bound by AGO1. Unexpectedly, a portion of miR158 in wildtype remains unmethylated and thus subject to uridylation and destabilization, suggesting that plants naturally utilize miRNA methylation to modulate miRNA accumulation. Our results suggest that the AGO1-containing RISC complex may undergo programming to reflect each bound miRNA, determining a defined, distinct decay destiny. In this analysis, we sequenced sRNAs from two hen1 mutant alleles in Arabidopsis and three hen1 alleles in rice. In Arabidopsis, the strong hen1-1 allele in the Landsberg erecta (Ler) ecotype is the first hen1 mutant and emerged from an enhancer screen in the hua1-1/hua2-1 background, and hen1-8 in the Columbia (Col) background is a weak allele. In rice, WAVY LEAF1 (WAF1) is the ortholog of Arabidopsis HEN1, and two mutant alleles waf1-1 and waf1-2 each bear a single-base substitution leading to a premature stop codon in the second exon and a non-functional splicing site of the fourth intron, respectively. We identified a third mutant allele of the rice HEN1 gene (Oshen1-3 from the Korean (POSTEC) rice T-DNA mutant population).

Project description:It is largely unknown how microRNAs (miRNAs) are degraded in vivo or whether active miRNA turnover is critical to specific developmental processes. Arabidopsis ARGONAUTE10 (AGO10) maintains stem cell homeostasis in meristems by repression of miR165/6, a conserved miRNA acting through AGO1. Here, we report that AGO10, through its binding to miR165/6, reduces miR165/6 accumulation by enhancing its decay. We show that AGO10 promotes the 3’ truncation of miR165/6 by SMALL RNA DEGRADING NUCLEASES (SDNs) and that AGO10-bound miR165/6 is more susceptible to degradation by SDN1 than AGO1-bound miR165/6. Our work uncovers a mechanism that governs the stability of miRNAs, establishes the importance of spatially controlled miRNA decay in stem cell homeostasis, and sheds light on the diversification and specialization of Argonaute proteins. Both biogenesis and degradation contribute to the steady-state levels of microRNAs (miRNAs). miRNA degradation in both plants and animals is associated with 3’ truncation and 3’ uridylation, and in Arabidopsis, the nucleotidyl transferase HEN1 SUPPRESSOR1 is responsible for 3’ uridylation. The SMALL RNA DEGRADING NUCLEASE (SDN) family of 3’ to 5 exonucleases degrades short RNAs in vitro and limits the accumulation of miRNAs in vivo, but it is not known whether SDNs are responsible for the accumulation of 3’ truncated miRNA species in vivo. In addition, there has not been an example of active miRNA turnover being critical to specific developmental processes. Arabidopsis ARGONAUTE10 (AGO10) maintains stem cell homeostasis in meristems by sequestration of miR165/6, a conserved miRNA acting through AGO1. Here, we report that AGO10, through binding to miR165/6, reduces miR165/6 accumulation by enhancing its degradation. We show that SDNs are responsible for the generation of 3’ truncated miRNAs in vivo and AGO10 promotes the 3’ truncation of miR165/6 by SDNs. Our work uncovers a novel mechanism of miRNA degradation mediated by an argonaute protein, which contrasts the known stabilizing effects of most AGO proteins on their associated miRNAs. This work, together with previous studies on AGO10, demonstrates that spatially regulated miRNA degradation underlies stem cell maintenance in plants.

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Differentiated Truncation and Uridylation Patterns of miRNAs in Arabidopsis and Rice hen1 Mutants Suggest miRNAs May Re-program RISC

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