Degradation of microRNAs by a family of exoribonucleases in Arabidopsis - PubMed (original) (raw)

Degradation of microRNAs by a family of exoribonucleases in Arabidopsis

Vanitharani Ramachandran et al. Science. 2008.

Erratum in

Abstract

microRNAs (miRNAs) play crucial roles in numerous developmental and metabolic processes in plants and animals. The steady-state levels of miRNAs need to be properly controlled to ensure normal development. Whereas the framework of miRNA biogenesis is established, factors involved in miRNA degradation remain unknown. Here, we show that a family of exoribonucleases encoded by the SMALL RNA DEGRADING NUCLEASE (SDN) genes degrades mature miRNAs in Arabidopsis. SDN1 acts specifically on single-stranded miRNAs in vitro and is sensitive to the 2'-O-methyl modification on the 3' terminal ribose of miRNAs. Simultaneous knockdown of three SDN genes in vivo results in elevated miRNA levels and pleiotropic developmental defects. Therefore, we have uncovered the enzymes that degrade miRNAs and demonstrated that miRNA turnover is crucial for plant development.

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Figures

Figure 1

Figure 1

Arabidopsis At3g50100 (SDN1) possesses 3’-to-5’ exonuclease activity on miRNAs. A, enzymatic activity assays on single-stranded miRNAs in vitro. RNA oligonucleotides were 5’-end labeled, incubated with buffer alone (1), purified GST (2), or purified GST-At3g50100 (3), and resolved on a denaturing polyacrylamide gel. miR173-me, a miR173 oligonucleotide containing a 2’-_O_-methyl group on the 3’ terminal ribose. B, enzymatic activity of GST-At3g50100 (GST-SDN1) on miR173 labeled at the 3’ end with 32pCp. miR173-32pCp was treated (+) or not (−) with phosphatase before incubation with GST-SDN1. The arrow indicates the position of the expected 15 nt product if SDN1 were to cleave the RNA between nt 8 and 9 from the 5’ end. The radioactivity at the bottom corresponds to the position of free nucleotides.

Figure 2

Figure 2

Substrate specificity of SDN1. A, RNA oligonucleotides ranging from 17 to 27 nt in length were 5’-end labeled and incubated with GST-SDN1. S, substrates alone; E, substrates + GST-SDN1. B, a 5’-end labeled, single-stranded DNA oligonucleotide (ssDNA) and a miR173/miR173* duplex labeled at the 5’ end of miR173 (dsRNA) were each incubated with buffer (S), GST (G), or GST-SDN1 (E). C, an in vitro transcribed and 5’-end labeled pre-miR167 was incubated with buffer (S), GST (G), or GST-SDN1 (E). An in vitro transcribed and 5’-end labeled AP1 RNA of approximately 300 nt was incubated with GST-SDN1 for 0, 30, and 60 min. D, miR173 or miR173 with 2 or 5 additional Us at the 3’ ends was each incubated with GST-SDN1 for 0, 10, or 30 min. E, effects of the 2’-_O_-methyl group on GST-SDN1 activity. Top panel: 5’-end labeled miR173 or 2’-_O_-methyl miR173 was incubated with increasing amounts of GST-SDN1 (numbers 0–5 represent 0 ng/μl, 0.33 ng/μl, 0.67 ng/μl, 1.33 ng/μl, 2.0 ng/μl, and 2.67 ng/μl of GST-SDN1, respectively). Bottom panel: miR173 or 2’-_O_-methyl miR173 was incubated with 0.67 ng/μl GST-SDN1 for the time specified periods (min). The arrow indicates a ~20 nt intermediate. Note that under low enzyme concentration, another intermediate of 9–10 nt was also present (in D and E). The bottom band corresponds to the final, 8–9 nt product.

Figure 3

Figure 3

Northern blot to detect the steady-state levels of seven miRNAs and an siRNA in mutants of SDN1 and related genes. The U6 blots serve as a loading control. The numbers below the blots indicate the relative abundance of the small RNAs in the different genotypes. 1, wild type; 2, sdn1–1; 3, the mutant in At3g50090; 4, sdn2–1; 5, sdn3–1; 6, sdn1–1 sdn2–1.

Figure 4

Figure 4

Effects of an amiRNA that targets SDN1 and three related genes. A, an sdn1–1 plant. B-F, amiRNA lines (in sdn1–1) with developmental defects of varying severity. B-D, type I plants. Arrowhead in C indicates a pin-like protrusion. E, a type II plant that has small, mildly serrated leaves. F, an early-flowering type III plant. G, accumulation of miR167 in wild type (Col), sdn1–1, and two individual type I amiRNA lines. The numbers below the gel images indicate the relative abundance of the miRNA. H-I, levels of SDN and AGO1 mRNAs as determined by RT-PCR (H) and realtime PCR (I) in the four genotypes in G. J, northern blotting to detect the amiRNA and endogenous small RNAs in pooled amiRNA lines. The numbers below the gel images indicate the relative abundance of the small RNAs. K, RT-PCR to detect SDN transcripts in wild type (Col), sdn1–1, and six pools of amiRNA lines. The “-RT” controls did not yield any products and were not shown. The three bands for SDN2 probably represent alternative transcripts since all three were missing in the “-RT” control.

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