PMC

Features of X. laevis oocyte Piwi-protein associated transcripts (PATs). (A) Histogram of JGI v.1.8 transcript enrichments (defined as: IP fpkm/total RNA fpkm) from RIP-seq libraries. (Inset) Venn diagram of the top 5% of enriched transcripts from XIWI and XILI RIP-seq libraries. (B) Validation of transcripts that have high RIP-seq enrichment by RT-qPCR analysis of transcripts from XIWI and XILI IPs; bysl and sdad1 are negative controls. Bars represent an average of two biological replicates. (C) Histogram of piRNA cluster transcript enrichments (defined as: IP fpkm/total RNA fpkm) from RIP-seq libraries. (D–G) XIWI and XILI RIP-seq libraries counted against JGI v.1.8 transcripts. Spearman's rank correlation denoted by rho (ρ). RIP-seq enrichments plotted against: (D) the number of perfectly mapping piRNAs; (E) transcript length; (F) transcript length for only transcripts with mapping ping-pong piRNA pairs; (G) and transcript expression level.

Distinctions between X. laevis oocyte XIWI and XILI piRNAs. (A) Coprecipitating RNAs from XIWI, XILI, and mock (preimmune rabbit IgG) IPs were separated on a 15% urea polyacrylamide gel and stained with SYBR Gold. (B) Histogram of XIWI and XILI piRNA strand lengths from the piRNA-seq library. (C) piRNAs mapped to X. laevis genome build 9.1 and counted against JGI v.1.8 transcripts (transcriptome) and piRNA cluster transcript models. (D) Base frequencies of all XIWI and XILI piRNAs as well as piRNAs with ping-pong signatures. (E) Percent of genomic mapping piRNAs with ping-pong signatures. (F) XIWI and XILI piRNAs mapping to Scaffold 23 in the X. laevis genome build 9.1, with a zoomed region from the dashed line box shown on the right panel. The piRNA cluster transcript models are denoted by dark blue rectangles above scaffold coordinates on the x-axis.

Characterization of XILI, homolog of XIWI, in X. laevis oocytes. (A) Phylogenetic tree of a selection of vertebrate Piwi proteins compared against the Drosophila Piwi proteins. The chicken homolog of PIWIL2/HILI was omitted because only a partial fragment of the protein sequence exists. Human Argonaute (Ago) proteins are used as an outgroup subclade to root the tree. (B) Western blot for XILI and XIWI in X. laevis stage 1–3 oocyte extract. (C) Western blot for XILI and XIWI in immuno-precipitates from XILI, XIWI, and preimmune rabbit IgG (mock precipitation). (D) Western blot for XILI and XIWI in immunodepleted X. laevis stage 1–3 oocyte extracts. (E) Western blot for XILI in a single X. laevis oocyte at the indicated stage of oogenesis. (F–I) Confocal imaging of XILI localization in a stage 1 X. laevis oocyte, with XILI and Tubulin patterns alone (F,G) and merged (H). Arrow points to the concentration of XILI in the Balbiani body. (I) Quantification of the XILI and Tubulin signals on a vertical plane that bisects the Balbiani body, supporting the XILI concentration in that organelle as well as in the germinal vesicle/nucleus of the oocyte. Scale bar represents 50 microns. (J) Confocal imaging of XILI localization in stage 2 and 3 oocytes.

Features of X. tropicalis oocyte PATs and comparison to X. laevis oocyte PATs. (A) Western blots of IPs from stage 3–4 X. tropicalis oocytes. (B) Left panel shows radiolabeled RNAs from different staged X. tropicalis oocytes irradiated with UV and then subjected to IPs of XIWI and XILI resolved on a denaturing polyacrylamide gel. Right panel shows an ethidium bromide-stained agarose gel of the RNA fragments associating with XIWI and XILI IPs from extracts of UV-irradiated and RNase T1-treated X. tropicalis stage 1–4 oocytes; and brackets indicate the region excised for RNA sequencing. (C) The proportions of genomic functional annotations of the XIWI and XILI CLIP-seq reads, poly(A) RNAs, and piRNAs from X. tropicalis stage1–4 oocytes. (D) Genome browser tracks of reads from CLIP-seq, mRNA-seq, and piRNA-seq for two top represented TEs and two developmentally important gene transcripts. (E,F) Scatterplots and Pearson correlation (R²) values comparing between the various CLIP-seq, mRNA-seq, and piRNA-seq libraries for gene transcripts (E) and TEs (F). (G) Scatterplot comparing the amount of enrichment for candidate genes and TEs tested in RIP experiments versus their RPM counts of the CLIP-seq data sets. (H) Venn diagrams and scatterplots showing strong commonality and positive correlation in enrichment levels for XIWI- and XILI-associated transcripts compared between X. laevis and X. tropicalis oocytes. CLIP counts and piRNAs are log₁₀ of reads per million, and fpkm is fragments per kilobase per million.

X. laevis PATs with high amounts of targeting piRNAs are degraded rapidly in oocyte extracts. (A) XIWI and XILI RIP-seq and piRNAseq libraries counted against UniGene transcripts. Specific transcripts denoted by color. (B) Degradation assays in X. laevis stage 1–3 oocyte extract. (C) Quantitated band intensities for degradation assay Northern blots. Sense nc3 transcript and antisense ncRNAgp transcript replicates where N = 4 and N = 5, respectively. Error bars denote the standard error of the mean (SEM). (D) XIWI and XILI piRNAs with perfect complementarity to ncRNAgp and nc3. (E) Western blot for molecular markers in X. laevis stage 1–3 oocyte extract (o.e.), cytosolic fractions (c.f.), and membrane fractions (m.f.). Trap-α marks endoplasmic reticulum membranes; hsp60 marks mitochondria; α-tubulin marks cytosol. (F) Degradation assay with nc3 sense strand in X. laevis stage 1–3 oocyte extract and fractionated cytosolic and membrane fractions. (G) Exogenous RNA association assay shows that a 3.2-kb-long RNA (encoding EMTB-3XGFP) is enriched in IPs with XIWI in a concentration-dependent and XIWI-dependent manner, since there is minimal RNA recovery in the IgG negative control. Box plots of experiments from three separate frog oocyte extracts. (H) The 3.2-kb-long RNA is more enriched in XIWI IPs compared to the 1.8-kb-long RNA (encoding EMTB-1XGFP), with much less distinction in XILI IPs. Box plots of experiments from four separate frog oocyte extracts.