Figure 5. CSR-1 Associates with Both Male- and Female-Specific Small RNAs in Males and Positively Regulates Spermiogenic Gene Expression (A) Left: fertility of WT,
csr-1, or
csr-1 alg-3;
alg-4 males at 20Cor 25C measured in crosses with
fog-2 females. Box and whisker plots represent a range of cross progeny from at least 15 independent crosses for each genotype. Right: in vitro activation of spermatids isolated from
csr-1,
csr-1 alg-3;
alg-4, orWT males grown at 20C or 25C. Bar graphs show the percent of spermatids that fail to activate (red), partially activate and form spikey projections (orange), or fully activate and form a pseudopod (yellow) (n > 300). (B) Confocal IF images of primary (1) spermatocytes (left) or spermatids (right) in dissected
alg-3/4 orWT germlines stained with antibodies against CSR-1 (green) and a nuclear pore protein (red). DNA was stained with DAPI (blue). White arrows denote chromatin domains where CSR-1 localizes. (C) Box and whisker plots indicate depletion of 26G-RNAs and 22G-RNAs antisense to ALG-3/4 targets in
alg-3/4 and
csr-1 males at 25C. Box and whisker plots to the right indicate enrichment of 22G-RNA and lack of enrichment of 26G-RNAs antisense to ALG-3/4 targets in the CSR-1 IP. (D) Analysis of mRNA expression and CSR-1 small RNA levels inWT males. Categories of gene expression are indicated below the bottom graph. ALG-3/4 targets as defined in Figure 1: positively regulated (green), negatively regulated (yellow), and unchanged (white). Male-germline-specific genes (blue) include many ALG-3/4 targets. Female-germline specific genes (red) are not targeted by ALG-3/4. Soma-specific mRNAs and 21U-RNAs are control sequences not enriched by CSR-1 IP. The number of genes in each category is indicated above the graphs. Top graph: mRNA expression as monitored by RNA-Seq in reads per million per kilobase (RPMK). Middle graph: small RNA levels in reads per million (RPM) per gene. Bottom graph: box and whisker plot indicating the enrichment or depletion in CSR-1 IP of small RNAs antisense to genes. Enrichment was calculated as the RPM ratio of FLAG::CSR-1 IP/(FLAG::CSR-1 IP + input). Dotted lines indicate 2-fold enrichment (upper) or depletion (lower). The plus sign (+) indicates the average enrichment value for all genes in the category. (E) qPCR analysis of FLAG::CSR-1 ChIP at genes targeted by ALG-3/4, CSR-1, or neither. ALG-3/4 negatively regulated targets are indicated in gray text. Data were normalized to
y47h10a.3, which is not targeted by ALG-3/4 or CSR-1. Data are represented as mean ± SEM.