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Degradation of unmethylated miRNA/miRNA*s by a DEDDy-type 3’ to 5’ exoribonuclease ATRIMMER2 in Arabidopsis

ABSTRACT: 3’ end methylation catalyzed by HUA ENHANCER1 (HEN1) is a crucial step of small RNA stabilization in plants, yet how unmethylated small RNAs undergo degradation remains largely unknown. Using a reverse genetic approach, we here show that ATRIMMER2 (ATRM2), a DEDDy-type 3’ to 5’ exoribonuclease, acts in the degradation of unmethylated miRNAs and miRNA*s in Arabidopsis. A loss-of-function mutations in ATRM2 partially suppress the morphological defects caused by HEN1 malfunction, with restored levels of a subset of miRNAs and receded expression of corresponding miRNA targets. Loss of ATRM2 has negligible effect on miRNA trimming, and further increase the fertility of hen1 heso1 urt1, a mutant with an almost complete abolishment of miRNA uridylation, indicating that ATRM2 may neither be involved in 3’ to 5’ trimming nor be the enzyme that specifically degrades uridylated miRNAs. Notably, the fold changes of miRNAs and their corresponding miRNA*s were significantly co-regulated in hen1 atrm2 as compared with those in hen1. Unexpectedly, we also observed a markedly increase of 3’ to 5’ trimming of several miRNA*s but not miRNAs in ATRM2 compromised backgrounds. These data suggest an action of ATRM2 on miRNA/miRNA* duplexes, and the existence of an unknown exoribonuclease for specific trimming of miRNA*. This asymmetric effect on miRNA/miRNA* is likely related to ARGONAUTE (AGO) proteins, which can distinguish miRNAs from miRNA*s. Finally, we show that ATRM2 colocalizes and physically interacts with ARGONAUTE1 (AGO1). Taken together, our results suggest that ATRM2 may be involved in the surveillance of unmethylated miRNA/miRNA* duplexes during the initiation step of RISC assembly.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE107070 | GEO | 2018/12/01

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).

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Degradation of unmethylated miRNA/miRNA*s by a DEDDy-type 3’ to 5’ exoribonuclease ATRIMMER2 in Arabidopsis

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