(A and B) Nomarski images of the lateral surfaces of adult wild type (wt) and ain-1(ku322);ain-2(tm1863) double mutant animals. wt worm had continuous alae (arrow), while the ain-1;ain-2 double mutant in the photo displays a severe defect in alae formation.
(C and D) Nomarski image of the vulvae of adult wt and ain-1(ku322);ain-2(tm1863) double mutant animals. The ain-1;ain-2 double mutant displayed a protruding vulva phenotype (arrow in D).
(E and F) Fluorescence images showing the expression of the seam cell specific marker SCM::GFP in wt and mutant adult worms. The ain-1; ain-2 double mutants displayed a drastic increase in seam cell numbers (F).
(G). Percentage of adult animals with alae formation defects. Genotype of each strain is indicated below each bar. Severe defect describes animals missing >50% of their alae including those missing alae. Weaker defect describes animals that lack < 50% of their alae.
(H) Percentage of adult animals with a protruding vulva (Pvul) mutant phenotype.
(I) Average numbers of SCM::GFP positive cells on one lateral side of adult animals. In adult ain-1(lf);ain-2(rf) double mutants, the seam cells expanded to a wider area and the intensity of GFP fluorescence was highly heterogeneous, similar to that in the lin-66(lf); daf-12(lf) double mutant (Morita and Han, 2006); unpublished data).
(J)Average number of SCM::GFP positive cells on one lateral side of late L1 larvae before L1 molting and late L2 larva before L2 molting.
n indicates the number of animals examined.

(A) Relative abundance of 130 annotated miRNAs in different samples. miRNA reads in each sample were normalized to the corresponding sequencing scale. Each horizontal bar within the column represents a single miRNA. Red intensity represents the square root transformed relative abundance of each miRNA. The correlation coefficient shown at the top was calculated using the CORREL function of Microsoft Excel.
(B) Percentages of miRNA, siRNA and 21U RNA in the 20–25nt length fractions of indicated samples.
(C) Hairpin precursor structures of mir-1 and two potential new miRNAs. The RNA structures represent the lowest free energy folding predicated by mfold (Mathews et al., 1999; Zuker, 2003). Mature miRNA sequences are capitalized and highlighted in green.

(A) Histogram of average percentile rank of enrichment after AIN-1 IP. Red shading indicates the genes (2833) that were significantly enriched after AIN-1 IP but not after control pre-immune serum IP (Experimental Procedures).
(B) Histogram of average percentile rank of enrichment after AIN-2::GFP IP. Red shading indicates the genes (1995) that were significantly enriched after AIN-2::GFP IP but not after the control GFP IP (Experimental Procedures).
(C) Venn diagram of number of enriched genes shared between those from the AIN-1 and AIN-2 IP.
(D) Pie charts displaying the genes of different classes in four gene pools. The distribution of different gene classes in AIN-1 or AIN-2 associated genes are essentially the same as that is in the total gene pool.

(A and B) Fluorescence images showing the expression of lin-28::GFP in L3 animals of genotypes indicated. While only 5% of the wt animals showed detectable GFP in hypodermal nuclei at the L3 stage (n=21), 95% of the ain-2(rf); ain-1 RNAi animals displayed detectable GFP signals in a few random hypodermal nuclei (arrowheads) at the same stage (n=21).
(C and D) Fluorescence images showing the expression of hbl-1::gfp in wt L4 animals of genotypes indicated. While no florescence could be detected in hypodermal nuclei in worms with mock RNAi (Panel C, n=20), prominent GFP signals were visible in most of the hypodermal nuclei (arrowheads) in 86% of the ain-2(rf); ain-1 RNAi mutant animals (Panel D, n=21).
(E) Northern blot of mir-48 and mir-84 miRNAs in wt and ain-1(lf),ain-2(rf) (labeled as ain-) L3 larvae. U6 snRNA was measured as the loading control. The levels of mature mir-48 and mir-84 miRNAs (arrow) were not significantly different between wt and ain-1(lf),ain-2(rf) samples. No accumulation of miRNA precursors (arrowhead) were observed in the ain-1(lf), ain-2(rf) sample compared to that in the wt sample.
(F) Protein sequence coverage of several proteins after MuDPIT analysis of the indicated immunoprecipitation (IP) results. Control IP 1 was the IP sample obtained using an anti-GFP antibody from wt worm lysate. Control IP 2 was the IP sample obtained using the anti-GFP antibody from the worm lysate of an ain-2 promoter::gfp expressing strain.
(G) Western blot using the anti-AIN-1 antibody. The lysates from worms expressing the indicated fusion proteins were immunoprecipitated using an anti-GFP antibody followed by western analysis.

(A) Fold of enrichment of mRNAs in the AIN-2::GFP IP sample and the control GFP IP sample. mRNA levels were determined by qRT-PCR and normalized to the internal eft-2 control mRNA. Fold enrichment of a given mRNA was determined by comparing the normalized concentration of this mRNA in the IP sample to the corresponding input worm lysate sample.
(B) Fold of enrichment of mRNAs in AIN-2::GFP IP using indicated RT primers. There was no significant difference between results using an oligo-dT primer and random hexamer primers.
(C) Fold of enrichment of mRNAs of indicated genes in AIN-2::GFP and control GFP IP from wt and lin-4(lf) animals. Random hexamers were used as RT primers. The enrichment of lin-14 was significantly decreased in lin-4(lf) compared to the wt background, while the enrichment of hbl-1 and lin-28 remained unchanged.
(D) Fold of enrichment of mRNAs of indicated genes in AIN-2::GFP at three development stages. An oligo-dT primer was used as the RT primer. npp-18 was used as the internal reference gene. lin-14 was enriched in L2 but not in L1 stage. This enrichment was further increased in L3 stage. lin-41 was enriched in L3 but not in L1 or L2 stage.
Error bars indicate standard deviations in the corresponding experiments.

(A) Fluorescence images showing that lin-45a 3′UTR is responsible for a negative regulation of its expression in vulval precursor cells at L3–L4 stage (arrows).
(B) Fluorescence images showing that lin-29 3′UTR received negative regulations at young adult stage hypoderm cells (arrows).
(C) Fluorescence images showing that lev-1 3′UTR received negative regulations at young adult ventral and dorsal cord neuron cells (arrows).
(D) Fluorescence images showing that ced-9 3′UTR received negative regulations at a few young adult head cells (arrows). The experimental design for co-expressing both GFP and DsRED reporters and the quantitative analysis of the results presented in (A–D) are also shown in Figure S4.