Figure 2. Aberrant production of 22G-RNAs from rRNAs in prg-1 mutants.

(A and B) Scatterplots displaying each non-coding RNA as a function of normalized log₂-transformed small RNA high-throughput sequencing reads in wild-type and prg-1(n4357) (A) or mut-16(pk710) (B) mutants (axes values are reverse transformed to reflect non-transformed values). rRNAs are circled. Libraries are from dissected distal gonads (n = 3 biological replicates for each strain). Solid lines above and below the y = x lines indicate 2 and −2 fold-changes.

(C) Small RNA read distribution across an rRNA locus. One representative of 3 biological replicates is shown for wild-type, prg-1(n4357), and mut-16(pk710).

(D) Antisense rRNA-derived small RNA size distribution in prg-1(n4357) mutants.

(E) Relative log₂-transformed levels of a 26S/28S rRNA-derived 22G-RNAs in wild-type animals and various mutants as determined by TaqMan qRT-PCR (normalized to miR-1). The y axis shows log₂-tranformed values. Error bars are mean ± SD (n = 3). Bonferroni-corrected p value range: 2.8 × 10ˆ5 − 0.0049 (two-sample t tests).

(F) Scatterplots displaying each small RNA feature colored by class as a function of normalized log₂-transformed small RNA reads in wild-type and mutant animals (axes are reverse transformed to reflect non-transformed values).

(G) Enrichment of rRNA-derived 22G-RNAs in FLAG::HRDE-1 co-immunoprecipitates relative to input cell lysates from wild-type and prg-1(n4357) mutants.

(H) Relative levels of a 26S/28S rRNA-derived 22G-RNA in wild-type and prg-1(tm872) mutant animals as determined by TaqMan qRT-PCR (normalized to miR-1). Error bars are mean ± SD (n = 3). The p value was calculated using a two-sample t test.

(I) Normalized log₂-transformed rRNA-derived 22G-RNA levels in wild-type and prg-1(n4357) and mut-16(pk710) mutant gonads. Error bars are mean ± SD (n = 3). p values were calculated using two-sample t tests followed by Bonferroni correction for multiple comparisons. The y axis shows log₂-transformed values.

(J) Pie chart displaying the proportion of all WAGO-class 22G-RNA reads from features classified as 22G-RNA hyperaccumulators (>250 reads on average and >5-fold increase in 22G-RNA levels in prg-1 mutants relative to wild-type) or non-hyperaccumulators. WAGO-class features were expanded to include the hyperaccumulators that are not classified as WAGO targets.

See also Figure S2 and Table S3.