1. Systematic Analysis of Tissue-Restricted miRISCs Reveals a Broad Role for MicroRNAs in Suppressing Basal Activity of the C. elegans Pathogen Response 
Brian A. Kudlow, Liang Zhang, Min Han 
Molecular Cell 
Volume 46, Issue 4, Pages (May 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1
Identification of miRISC-Associated mRNAs in Intestine and Muscle
(A) Total number of testable and AIN-2-associated transcripts in IPs from three different transgenic lines. Transcripts were considered testable if at least two biological replicates yielded microarray signals above a threshold value for both the Total RNA and IP RNA sample. mRNAs were considered enriched if their mean percentile rank was significantly (p < 0.01 for asynchronous cultures and p < for L4-synchronized cultures) above average.
(B) Bar graph illustrating the extent of overlap between transcripts from different IP samples (Actual) compared to the overlap expected by random chance (Expected). Unexpectedly high numbers of common transcripts were found in AIN-2 IP samples from both muscle and intestine. The GFP-only negative control represents samples from Pain-2::gfp transgenic line. Expected values and p values were calculated according to the hypergeometric distribution.
See also Figure S1, Table S1, and Documents S1, S2, S3, and S4.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 AIN-2-Associated mRNAs Are Enriched for miRNA Seed Matches
(A) Bar graph showing the average number of perfect 7-mer (nt 2–8) seed matches to known C. elegans miRNAs per 1000 nt of 3′ UTR sequence (from Jan et al., 2011) associated with mRNAs in each data set. The “Fold enrichment” represents the ratio of the seed match frequency in IP mRNAs compared to the corresponding set of testable mRNAs.
(B) Bar graph showing relative 7-mer (nt 2–8) seed match frequency in each set of IP mRNAs relative to the corresponding set of testable mRNAs for the indicated sets of miRNAs. “All miRNAs” indicates all annotated C. elegans miRNAs. For each percentile cutoff, only miRNAs within the indicated percentile of detectable miRNAs, by raw read count, in the given IP sample were considered (see analysis in Figure 3). Statistical analysis of enrichment can be found in Table S3.
See also Figure S2 and Tables S2 and S3.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Tissue-Specific AIN-2 IP Identifies Intestine-Enriched miRNAs
(A) Bar graph showing miRNAs that are significantly (p < 0.01) enriched in the intestine AIN-2 IP compared to total RNA. miR-1 is known to be specifically expressed in muscle and is shown as a control. Error bars represent the SEM. p values were computed by two-tailed paired t test.
(B) Statistical data showing the high-level and tissue-specific enrichment of two validated targets (Simon et al., 2008) of miR-1 in the IP samples from the muscle. Transcripts were considered to be enriched if their mean percent ranks were reproducibly (p < 0.01) above average. ND, not detectable.
(C) Bar graph representing enrichment of perfect 7-mer seed matches, compared to related scrambled controls (see the Supplemental Experimental Procedures) for miRNA families with at least one member showing at least 2-fold (p < 0.01) enrichment in the muscle AIN-2 IPs. miRNAs are listed from left to right according to decreasing raw read count in the muscle AIN-2 IPs. The ubiquitous and highly expressed miR-51 family is shown as a positive control. The neuron-specific lsy-6 miRNA (Johnston and Hobert, 2003) is shown as a negative control. Numbers above the bars indicate the ratio of enrichment of the actual seed compared to the scrambled seeds.
(D) Bar graph representing enrichment of perfect 7-mer seed matches, compared to related scrambled controls (see the Supplemental Experimental Procedures) for miRNA families with at least one member showing at least 2-fold (p < 0.01) enrichment in the intestine AIN-2 IPs. miRNAs are listed from left to right according to decreasing raw read count in the intestine AIN-2 IPs. The ubiquitous and highly expressed miR-51 family is shown as a positive control. The neuron-specific lsy-6 miRNA (Johnston and Hobert, 2003) is shown as a negative control. miR-1 seed matches among intestine AIN-2 IP mRNAs are shown for reference. Numbers above the bars indicate the ratio of enrichment of the actual seed compared to the scrambled seeds.
See also Figure S3 and Tables S4 and S5 for additional analysis of miRNA seed matches in tissue-specific IP mRNAs.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Pathogen-Responsive mRNAs Are Overrepresented among the Transcripts Associated with Intestinal, but Not Muscle miRISCs, and Many Are Upregulated in dcr-1(lf) and ain-1/2 (lf) Mutants
(A) AIN-associated mRNAs, in particular those encoded by pathogen-responsive genes, are often upregulated in dcr-1(lf) mutants. Bar graph showing the percent of AIN-associated mRNAs within a given class that were found to be upregulated in dcr-1(lf). DCR-1-regulated genes were identified in a previous study (Welker et al., 2007). Expected values and p values were calculated according to the hypergeometric distribution.
(B) Quantitative-RT-PCR showing that inactivation of both ain-1 and ain-2 results in the upregulation of several mRNAs that were found in the intestinal miRISC. DCR-1-regulated and infection-induced genes were identified in previous studies (Bolz et al., 2010; Shapira et al., 2006; Troemel et al., 2006; Welker et al., 2007; Wong et al., 2007). Data represents mean ± SEM; p value computed by paired t test from four separate biological replicates.
(C) Top: Quantitative-RT-PCR showing that both tsp-1 and tsp-2 are upregulated in a ain-2(rf);ain-1(lf) double mutant. However, tsp-1 shows significantly higher (3.3-fold ± 0.5-fold, p = 5.0 × 10−5) upregulation than tsp-2. Graph represents mean ± SEM p value computed by paired t test from six separate biological replicates. Middle: Schematic representation of the tsp-1/tsp-2 operon. SL2, spliced leader 2. Bottom: tsp-1 mRNA, but not tsp-2 mRNA, was enriched in two intestinal miRISC IPs, but not in control IPs. ND, not detectable.
See also Figure S4 and Table S6.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Reduced miRISC Activity Enhances Survival on P. aeruginosa likely through a Function Parallel to the PMK-1 and DAF-2/DAF-16 Pathways
(A) Survival on a lawn of PA14 P. aeruginosa of three ain-1(lf) alleles (ku322, tm3681 and ku425) and an ain-2(lf) on lawns of PA14 P. aeruginosa was compared to wild-type (N2) worms.
(B) Number of viable progeny produced per animal at 25°C. For each genotype at least ten animals were scored. Data represent mean ± SD; p values were computed using a two-tailed t test.
(C) Some targets of both the DAF-2/DAF-16 and PMK-1 signal transduction pathways are associated with the intestinal miRISC. Expected values and p values were computed according to the hypergeometric distribution.
(D) Quantitative-RT-PCR shows that miRISC-regulated pathogen-response genes (MRPRs) can be induced after exposure to PA14 P. aeruginosa in both pmk-1(lf) and daf-16(lf) mutants. For each mutant, data represents the relative mRNA level (log 2) in worms exposed to PA14 compared to worms exposed to OP50. Note that F08G5.6 and sod-3 are known targets of the PMK-1 (Troemel et al., 2006) and DAF-2/DAF-16 (Murphy et al., 2003) pathways, respectively.
(E) Survival of daf-16(lf) and pmk-1(lf) strains alone and in combination with ain-1(lf) on a lawn of PA14 P. aeruginosa. Survival curve data is representative of multiple experiments. Both ain-1(lf) and daf-16(lf);ain-1(lf) showed significantly enhanced survival compared to wild-type and daf-16(lf) worms (p < 1.0 × 10−7). Both pmk-1(lf) and pmk-1(lf);ain-1(lf) showed significantly reduced survival compared to wild-type worms (p < 1.0 × 10−10). p values for survival curves were calculated with a log-rank test.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Identification and Analysis of miRNAs Involved in the Pathogen Response
(A) Bar graph showing the fold enrichment (log 2) of miRNAs in the intestinal miRISC of L4-synchronized worms. Only miRNAs that were not identified in intestine miRISCs in asynchronous cultures are shown (see Figure 2). miR-243 (intestine-enriched), miR-1 (muscle-specific [Simon et al., 2008]), and lsy-6 (neuron-specific [Johnston and Hobert, 2003]) are shown as controls. Data presented as mean ± SEM from four separate biological replicates. p value, by pairwise t test, is < 0.01 for all values shown, except lsy-6 in which p = 0.02.
(B) Dynamic changes in miRISC-associated miRNAs upon infection. Bar graph shows relative abundance (log 2) of miRNAs showing significant changes in either Total or in the miRISC IPs in L4 worms fed PA14 versus OP50. Values with error bars (SEM) have a p value < 0.01 as computed by a paired t test from four separate biological replicates.
(C) Heat map showing quantitative RT-PCR analysis of MRPR genes' expression in several strains deficient for specific miRNAs. n, number of independent biological replicates. p values were computed by a paired t test with culture-matched wild-type controls.
(D) Survival of mir-70(lf), mir-243(lf), mir-252(lf);mir-251(lf), and mir-253(lf) mutants on a lawn of PA14 P. aeruginosa. Data is representative of multiple experiments. p values were calculated with a log-rank test.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions