1. Volume 28, Issue 4, Pages 598-613 (November 2007)
Systematic Identification of C. elegans miRISC Proteins, miRNAs, and mRNA Targets by Their Interactions with GW182 Proteins AIN-1 and AIN-2 
Liang Zhang, Lei Ding, Tom H. Cheung, Meng-Qiu Dong, Jun Chen, Aileen K. Sewell, Xuedong Liu, John R. Yates, Min Han 
Molecular Cell 
Volume 28, Issue 4, Pages (November 2007)
DOI: /j.molcel
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
The ain-1(lf);ain-2(lf) Double Mutant Displays Robust Vulval and Seam-Cell Defects
(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heterogeneous, similar to that in the lin-66(lf); daf-12(lf) double mutant (Morita and Han, 2006; K. Morita and M.H., unpublished data).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, number of animals examined.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
AIN-1 and AIN-2 Associate with Both ALG-1 and ALG-2 In Vivo and Negatively Regulate the Expression of lin-28::gfp and hbl-1::gfp Reporter Genes without Affecting miRNA Biogenesis
(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 ([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 ([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) was 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.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3
AIN-1 and AIN-2 Associate with Most of the Known miRNAs and Nine Likely New miRNAs, but Not with siRNAs or 21U RNAs
(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 20–25 nt 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.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4
AIN-2 Complexes Contain Known Targets of miRNAs, and the Enrichment of These mRNAs Correlates with the Expression of Corresponding miRNAs
(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.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5
A Large Number of Potential miRNA Targets Was Identified through Their Association with AIN-1 and AIN-2
(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 preimmune 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 the 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 is essentially the same as that in the total gene pool.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6
3′UTR of lin-45a, lin-29, lev-1, and ced-9 Mediates Negative Regulations of Their Expression
(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 coexpressing both GFP and DsRED reporters and the quantitative analysis of the results presented in (A)–(D) are also shown in Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2007 Elsevier Inc. Terms and Conditions