1. DNA Methylation Mediated by a MicroRNA Pathway
Liang Wu, Huanyu Zhou, Qingqing Zhang, Jianguang Zhang, Fangrui Ni, Chang Liu, Yijun Qi 
Molecular Cell 
Volume 38, Issue 3, Pages (May 2010)
DOI: /j.molcel
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2. Figure 1 A Class of DCL3-Dependent LmiRNAs
(A) Size distribution of sequenced sRNAs isolated from wild-type rice (WT), DCL1IR-2, dcl3a-17, and rdr2-2 lines.
(B) Relative abundances of canonical miRNAs (cmiRNAs) and long miRNAs (lmiRNAs) in wild-type rice (WT), DCL1IR-2, dcl3a-17, and rdr2-2 lines. The relative abundance of each class of miRNAs is represented by its normalized RPMs.
(C) Detection of miRNAs and two siRNA clusters in wild-type rice (WT), DCL1IR, dcl3a, and rdr2 RNAi lines. The length and the 5′-terminal nucleotide of the sRNAs are shown in the parentheses. The northern blots were stripped and reprobed multiple times. U6 was probed and used as loading control. The positions of RNA size markers, electrophoresed in parallel, are shown to the right of the blots.
See Figure S1 for the characterization of dcl3a and rdr2 RNAi lines, Table S1 for the list of lmiRNAs, and Table S2 for the relative abundances of individual miRNAs in the RNAi lines determined by Illumina deep sequencing.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Processing of Dual-Coding miRNA Precursors by Coordinated Activities of DCL1 and DCL3
Models for processing of representative dual-coding miRNA precursors are shown (panels on the left). Northern blot analyses (panels on the right) were performed to examine the production of the cmiRNAs and lmiRNAs in wild-type rice (WT) and different RNAi lines as indicated. The blots were stripped and reprobed multiple times. U6 was probed and used as loading control. The positions of RNA size markers, electrophoresed in parallel, are shown to the right of the blots. See Figure S2 for the sequences and secondary structures of the miRNA precursors.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
CmiRNAs and LmiRNAs Are Sorted into AGO1 and AGO4 Clade Proteins, Respectively
(A) The AGO4 clade complexes (AGO4a, AGO4b, and AGO16) were immunopurified by using peptide-specific antibodies and were separated on 10% SDS-PAGE (upper panels). Preimmune antisera were used for the control purifications. The proteins were visualized by silver staining. The positions of protein size markers, electrophoresed in parallel, are shown to the left of each gel. The AGO protein bands are indicated by solid arrowheads. sRNAs were extracted from each AGO complex, analyzed by denaturing polyacrylamide gels, and visualized by SYBR Gold staining (lower panels). The positions of RNA size markers are shown to the left.
(B) The relative frequency of each 5′-terminal nucleotide of sRNAs in total RNA (Total) and those bound by AGO1 and AGO4 clade proteins.
(C) Relative abundances of cmiRNAs and lmiRNAs in AGO1 and AGO4 clade proteins. Relative abundances of cmiRNAs and lmiRNAs in AGO1 and AGO4 clade proteins were assessed by calculating enrichment or depletion in the AGO1 and AGO4 immunoprecipitates relative to the total extract.
(D) Detection of miRNAs and two siRNA clusters in total extract (Total) and AGO1 and AGO4 clade proteins as indicated. The length and the 5′-terminal nucleotide of the sRNAs are shown in the parentheses. The northern blots were stripped and reprobed multiple times. The positions of RNA size markers, electrophoresed in parallel, are shown to the right of the blots.
(E) Immunopurified AGO4a and AGO4b were incubated with single-stranded 32P-labeled 21 and 24 nt siRNAs bearing the indicated 5′-terminal nucleotides. Mixtures were irradiated with UV and resolved by 10% SDS-PAGE. Gels with shorter exposure (middle panels) are shown to indicate that comparable amounts of siRNAs were added into each reaction. Silver-stained gels (bottom panels) are shown as controls for the proteins used in the crosslinking reactions.
See Figure S3 for the confirmation of purified AGO4 clade proteins by mass spectrometry and Table S3 for relative abundances of individual cmiRNAs and lmiRNAs in AGO1 and AGO4 clade proteins.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Size of a Small RNA Is Not a Determinant for AGO Loading
(A) Immunopurified AGO1 and AGO4 clade proteins were incubated with single-stranded 32P-labeled 21 and 24 nt siRNAs bearing the indicated 5′-terminal nucleotides. Mixtures were irradiated with UV and resolved by 10% SDS-PAGE. The crosslinked products are indicated by solid arrowheads. Gels with shorter exposure (middle panels) are shown to indicate that comparable amounts of siRNAs were added into each reaction. Silver-stained gels (bottom panels) are shown as controls for the proteins used in the crosslinking reactions.
(B) Relative abundances of Arabidopsis miR163 in total extract (Total), AGO1, and AGO4 complexes. The relative abundance of miR163 is represented by its normalized RPMs.
(C) Detection of Arabidopsis miR163 and a cluster of heterochromatic siRNAs (AtREP2) in total RNAs isolated from WT plants (La-er and Col-0), dcl1-7 and dcl3-1 mutants, and in AGO1 and AGO4 immunoprecipitates. The northern blots were stripped and reprobed. The positions of RNA size markers, electrophoresed in parallel, are shown to the right of the blots.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 miR1873 Directs DNA Methylation at Its Own Locus
(A) Analysis of DNA methylation at miR1873 locus in WT rice by bisulfite sequencing. Sequencing data were analyzed by using Kismeth software ( The colored lines above the x axis show the percent methylation at individual cytosine sites. The colored lines below the x axis indicate the positions of cytosine sites. The regions corresponding to mature miR1873 and miR1873∗ are indicated by red and blue horizontal bars, respectively.
(B) DNA methylation status at the miR1873 locus in the indicated RNAi lines. The 5′-terminal nucleotide of miR1873 or miR1873∗ is set as position 1. Percent methylations of individual cytosine sites located in ±25 nt of miR1873 or miR1873∗ are shown. Cytosine sites within miR1873 and miR1873∗ regions are highlighted.
(C) DNA methylation status at two siRNA-producing loci, OsCluster1 and OsCluster 2, in WT rice, dcl3a-17, and rdr2-2 lines. Presented is overall percent methylation of cytosine sites in different sequence contexts.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
LmiRNAs Direct DNA Methylation at Target Genes and Regulate Their Expression
(A) Analysis of DNA methylation status at Os06g38480 (a target of miR1863) in WT rice by bisulfite sequencing. Sequencing data were analyzed by using Kismeth software ( The colored lines above the x axis show the percent methylation at individual cytosine sites. The colored lines below the x axis indicate the positions of cytosine sites. The region corresponding to the miR1863 target site is indicated by a red bar.
(B) DNA methylation status at Os06g38480 in the indicated RNAi lines. The first nucleotide of the target site is set as position 1. Percent methylation of individual cytosine sites located in ±25 nt of the target site are shown. Cytosine sites within the target site are highlighted.
(C) Relative expression levels of Os06g38480 and Os03g02010 in the indicated RNAi lines. The expression levels were normalized using the signal from the GAPDH gene. The average (± standard deviation) values from three biological repeats of quantitative reverse transcription PCR are shown.
See Figure S5 for more examples of lmiRNA-directed methyation at target genes.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
A Model for Two Distinct miRNA Pathways that Act at the mRNA and Chromatin Levels, Respectively
Our data from rice indicate that there are two distinct miRNA pathways that operate at mRNA and chromatin levels, respectively. In (A), canonical miRNAs (cmiRNAs) are generated by DCL1 and specifically loaded into AGO1 clade proteins to form effector complexes to direct the cleavage of their target mRNAs. In (B), long miRNAs (lmiRNAs) are processed by DCL3 and sorted into AGO4 clade proteins. LmiRNAs bound to AGO4 proteins interact with nascent transcripts transcribed from their own loci or target genes, thereby recruiting de novo cytosine methyltransferase DRM2 to methylate the adjacent DNA.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions