(A) Northern blot showing that 21UR-1 is not detected in RNA (40 μg) isolated from two independent piwi double-mutants: piwi(n4357; n4358) and piwi(n4503; nDf57), whereas miR-52 is expressed in both mutants (same blot re-probed). Antisense DNA probes were used for 21UR-1 and miR-52. A U6 northern blot is shown as loading control. piwi(n4357; n4358) is an abbreviation for prg-1(n4357); prg-2( n4358). piwi(n4503; nDf57) is an abbreviation for prg-1(n4503); prg-2( nDf57).
(B) High-throughput sequencing reveals that the expression of many 21U-RNAs is dramatically reduced in piwi mutants. 21U-RNAs cloned from 5′ dependent wild-type and piwi(n4357; n4358) mutant libraries. Frequencies are shown for wild-type (blue) and piwi mutant (red) for the 400 most abundant 21U-RNAs in wild-type, plotted in the order of their wild-type frequency. Read frequencies were obtained by dividing the number of reads for a given 21U-RNA by the total number of reads from the same library (left-hand y-axis). The corresponding absolute number of reads are indicated in the right-hand y-axes. 21U-RNAs for which frequencies are shown include the 21U-RNA with most reads in the piwi mutant library (21UR-3224), which was sequenced 8 times in the piwi mutant and 2,127 times in wild-type.
(C) Expression of a 23 nucleotide antisense RNA (siR23-69) and a 26 nucleotide antisense RNA (siR26-263) is not affected in piwi mutants (northern blotting, 40 μg total RNA, antisense DNA probes).
(D) Immunoprecipitation followed by RT-PCR for 21UR-5101 reveals that 21U-RNAs are associated with PRG-1 in C. elegans extracts.
(E) Quantitative RT-PCR of seven 21U-RNAs demonstrates that Piwi is not essential for 21U-RNA biogenesis. Total RNA was extracted from 12 hour adult C. elegans. Expression levels shown are relative to levels in wild-type RNA. miR-52 expression was used as an internal control. Data are from three independent biological replicates. Error bars represent standard error of the mean.

(A) 21UR-1 northern blotting of total RNA of wild-type and mutant young adult C. elegans. In the case of alg-1; alg-2(RNAi), alg-1 mutant L1 larvae were transferred to alg-2 RNAi feeding plates and young adult animals were harvested. A U6 northern blot is shown as loading control. See Table S1 in Supplemental Data for quantification of these results.
(B) 21UR-1 northern blotting of total RNA of wild-type and mutant young adult C. elegans. dcr-1 mutant animals used were homozygous animals derived from heterozygous mothers. To test for loss of DCR-1 activity in dcr-1 mutants, let-7 miRNA and pre-miRNA is shown. A U6 northern blot is shown as loading control.
(C) Distribution of 21U-RNAs on chromosome IV as detected by high-throughput sequencing of 5′ dependent wild-type, piwi(n4357; n4358), dcr-1 and mut-7 mutant libraries. Frequencies for a given 21U-RNA and locus were obtained by correcting the number of reads for multiple alignments and dividing by the total number of reads from the same library. Cumulative frequencies were plotted for non-overlapping 100 kb windows along chromosome IV.

(A) Profile of 21U-R1 expression during development. E, embryo. L1-L4, larval stages 1-4. YA, 12-hour adult. A, 24-48-hour adult. A U6 northern blot is shown as loading control.
(B) Quantitative RT-PCR of 21UR-5101. miR-52 expression was used as an internal control. Data are from three independent biological replicates. Error bars represent standard error of the mean.
(C) Quantitative RT-PCR of prg-1 mRNA. Actin mRNA was used as an internal control. Data are from three independent biological replicates. Error bars represent standard error of the mean.
(D) Northern blot showing that piRNA expression is restricted to the male and female germline. glp-1(gf), glp-1(lf), glp-4, fem-1 and fem-3 L1 larvae were grown to 12-hour adult stage at 25°C. 20 μg of total RNA was loaded in each lane. U6 and miR-52 northern blots are shown as loading controls.
(E) Quantitative RT-PCR of prg-1 mRNA. Actin mRNA was used as an internal control.

(A) Diagram of the genomic structure of the two most common DNA transposons in C. elegans, Tc1 and Tc3. Tc1 and Tc3 are flanked by inverted repeats and encode a single spliced transcript for transposase. bp, base pairs. n, number of copies in the wild-type strain N2.
(B) Distribution of Tc1 and Tc3 transposons in the C. elegans genome.
(C), (D) Quantitative RT-PCR of Tc1 or Tc3 transposase mRNA. As the genomic copies of Tc1 and Tc3 have minor sequence variations, the number of transposon loci amplified by each qRT-PCR primer pair are shown (n). Actin mRNA was used as an internal control. Expression levels shown are relative to levels from wild-type RNA. Data are from three independent biological replicates. Error bars represent standard error of the mean.

(A) piRNA expression is independent of MUT-7. 21UR-1 northern blotting of total RNA of wild-type and mutant young adult C. elegans. Total RNA is shown as loading control (GelStar).
(B) Tc3 associated small RNAs are absent in piwi mutants. Small RNAs mapping to the loci of Tc3 (top) and Tc1 (bottom) on chromosome I as identified by high-throughput sequencing of 5′ independent wild-type (left) and piwi mutant (right) libraries. Inverted repeat and exon sequences are indicated in green and pink respectively. The number of aligned sequence reads (blue) and number of aligned unique sequences (red) were plotted for each base pair position, with the top and bottom graph in each panel corresponding to the antisense and sense strand relative to the transposase transcript. Read and sequence counts were corrected for multiple alignments to the genome. The total number of reads from wild-type and piwi mutant libraries were comparable (2,963,895 and 3,017,027 of reads with perfect matches to the reference genome respectively).
(C) Tc3 transposase antisense siRNAs are dramatically reduced in piwi mutants. RNase protection assay using sense fragments of Tc1 and Tc3 transposase. Sense siRNAs were not detected above background levels using this assay (see Figure S4 in Supplemental Data). U6 was used as an internal control, but its concentration had to be titrated down due to some interference with small RNA detection (Figure S5).

(A) Small RNAs mapping to the opposite strand of nearby 21U-RNA loci show a preference for locations downstream of the 21U-RNA locus and were reduced in the piwi mutant (right) as compared to wild-type (left). Proximate small RNAs on the same strand as the 21U-RNA were also reduced in the piwi mutant (Figure S7). (Bottom) Rows correspond to 6,021 21U-RNA loci, ordered by genomic position with colors representing different chromosomes. For a given row (21U-RNA locus) dots correspond to the relative position of nearby antisense small RNAs as defined by the distance of the 5′ end of the cloned small RNA relative to the 21U-RNA 5′ end. (Top) Shown is the frequency of distances between 5′ ends of 21U-RNAs and antisense small RNAs. Frequencies were based on the normalized number of loci for 5′ unique sequences.
(B) A speculative model of the role of Piwi in Tc3 silencing. TGS, transcriptional gene silencing. PTGS, post-transcriptional gene silencing. See Discussion for an explanation.