Download Supporting Information Legends Supporting Figure 1. Amino acid

Supporting Information Legends
Supporting
Figure
1.
Amino
acid
sequence
alignment
between
Chlamydomonas AGO2 and AGO3. Red boxes indicate the four residues of the
catalytic tetrad.
Supporting Figure 2. Analyses of the mutated AGO2 gene structure.
(A) Schematic diagrams of the AGO2 and the mutated AGO2 genes. The first
half of the AGO2 genes is indicated. The black horizontal lines above or below
the AGO2 diagrams correspond to the regions amplified by genomic PCR. The
location of the AGO2 5th intron probe is indicated with double lines. PstI
restriction sites are indicated as P. (B) Genomic PCR of the indicated strains.
(C) Southern blotting of the indicated strains. To detect the intact and the
mutated AGO2 fragments, the AGO2 5th intron was used as a probe.
Supporting Figure 3. Expression of epitope tagged-AGO3 in the ago3-1
background results in the rescue of miR9897-3p-mediated translational
repression of gluc(9-12 mm). Each panel is representative of three independent
experiments. (A) Immunoblotting of the gluc protein in the indicated strains.
Gluc(9–12 mm)-2×ago3-1(AGO3)-2 was generated by a genetic cross between
Gluc(9–12 mm)-2 and ago3-1(AGO3)-2. This strain expresses the gluc(9–12
mm) transgene and tagged-AGO3 in the ago3-1 background. (B) Northern
blotting of gluc mRNA in the indicated strains. (C) Immunoblotting of epitope
tagged-AGO3. Cell lysate of Gluc(9–12 mm)-2×ago3-1(AGO3)-2 was incubated
with FLAG-M2 beads and, after immunoprecipitation, the beads-associated
tagged-AGO3 was eluted by competition with 3×FLAG peptides. The lysate
before beads incubation, the lysate after beads incubation, and the eluted
samples were analyzed.
Supporting Figure 4. Photoheterotrophic growth and TOC1 RNA accumulation
in the wild type and AGO mutant strains. Each panel is representative of three
independent experiments. (A) Photoheterotrophic growth curve of the indicated
strains. Each time point represents the mean of six replicates (three independent
experiments) +/− SD. (B) TOC1 sRNA abundance in the indicated strains,
calculated as reads per million (RPM). (C) Northern blotting of TOC1 transcripts
in the indicated strains.
Supporting Figure 5. Accumulation of miR B and its endogenous target, the
Cre17.g697550 transcript. (A) Abundance of miR B, including 3’ length variants,
in the indicated strains expressed as reads per million (RPM). (B)
Quantitative-RT-PCR analysis of Cre17.g697550 in the indicated strains.
Cre17.g697550 transcript levels were normalized to those of CBLP, then the
value corresponding to the Gluc(1×) strain was set to one and other values were
adjusted accordingly. Data indicated is the average  SD of three independent
experiments.
Supporting Table 1. Comparison of the AGO2 and AGO3 expression levels.
Supporting Table 2. Analysis of small RNA libraries.