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. 2004 Jan 27;101(4):1093-8.
doi: 10.1073/pnas.0307969100. Epub 2004 Jan 13.

The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation

Meng-Hsuan Han  1 , Saiprasad Goud, Liang Song, Nina Fedoroff
Affiliations

The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation

Meng-Hsuan Han et al. Proc Natl Acad Sci U S A. .

Abstract

The Arabidopsis HYL1 gene encodes a nuclear double-stranded RNA-binding protein. A knockout mutation of the hyl1 gene is recessive and pleiotropic, causing developmental abnormalities, increasing sensitivity to abscisic acid, and reducing sensitivity to auxin and cytokinin. We report that levels of several microRNAs (miRNAs; miR159, -167, and -171) are reduced in homozygous mutant plants, and levels of two of three tested target mRNAs are elevated. Conversely, the miRNA levels are elevated in plants expressing a HYL1 cDNA from a strong promoter, and the corresponding target RNAs are reduced. These changes result from alterations in the stability of the target RNAs. However, double-stranded RNA-induced posttranscriptional gene silencing is unaffected by the hyl1 mutation. One-third to one-half of the cellular HYL1 protein is in a macromolecular complex, and a GFP-HYL1 fusion protein is found predominantly in the nucleus, although it is observed in both nucleus and cytoplasm in some cells. Within nuclei, HYL1 is associated with subnuclear bodies and ring-like structures. These observations provide evidence that the HYL1 protein is part of a nuclear macromolecular complex that is involved in miRNA-mediated gene regulation. Because hyl1 mutants show marked abnormalities in hormone responses, these results further suggest that miRNA-mediated changes in mRNA stability play a vital role in plant hormone signaling.

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Figures

Fig. 1.
Fig. 1.
miRNAs and predicted target mRNAs in 4-week-old wild-type, hyl1, hen1-1, and 35S::HYL1 plants (lanes 1 and 3 are from independent transformants). (A) RNA was fractionated on a denaturing 15% polyacrylamide gel, transferred to a membrane, and probed with 32P-labeled antisense oligonucleotides for the indicated miRNAs. (B) RNA was fractionated on a 1.2% agarose gel, transferred to a nylon membrane, and probed with 32P-labeled PCR-amplified fragments of the target RNAs indicated on the right. After hybridization and band quantification, the membranes used in A and B were stripped and rehybridized with additional probes. (C) RT-PCR analysis of DCL1 expression in wild-type, hyl1, and hen1-1 plants. The numbers below the blots are the ratio of the amount of the indicated RNA to the amount of RNA detected in wild-type plants.
Fig. 2.
Fig. 2.
Stability of target RNAs in wild-type, hyl1, and 35S::HYL1 (transformant 1 in Fig. 1) plants. (A) RT-PCR amplification of several target RNAs at various intervals after addition of 100 μg/ml cordycepin. (B) The results of several experiments such as those shown in A were quantified and plotted as a percentage of the initial value. Average values obtained with wild-type (wt) plants, hyl1 mutants, and 35S::HYL1 overexpressers are represented by diamonds, squares, and triangles, respectively.
Fig. 3.
Fig. 3.
PTGS in wild-type and hyl1 protoplasts. (A) Protoplasts from wild-type plants were transfected with H2O (mock-transfected), a 35S::LUC plasmid, and both the 35S::LUC and a LUC-dsRNA. After 12–16 h at 22°C, the transfected protoplasts were lysed, and LUC activity was measured (the lysis buffer was used as a control for the LUC assay). (B) The experiment was done by using hyl1 protoplasts transfected with both 35S::LUC and 35S::GUS genes; the protoplasts were then subdivided, and the indicated dsRNAs were added to aliquots, which were then incubated and assayed for both activities.
Fig. 4.
Fig. 4.
Subcellular distribution of HYL1-GFP fluorescence. (A) Nuclear, uniform. (B) Nuclear and cytoplasmic. (C) Ring and bodies. (D) Ring. Blue arrows indicate the nucleus, white arrows designate HYL1-containing rings, and white arrowheads indicate the HYL1-containing nuclear bodies. The red structures are chloroplasts.
Fig. 5.
Fig. 5.
Blue native gel electrophoretic analysis of HYL1. Total protein extract from wild-type plants was fractionated as described in Materials and Methods, transferred to a poly(vinylidene difluoride) membrane, and probed with anti-HYL1 antibody.
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