Cbp80 is needed for the expression of piRNA components and piRNAs

Abstract

Cap binding protein 80 (Cbp80) is the larger subunit of the nuclear cap-binding complex (nCBC), which is known to play important roles in nuclear mRNA processing, export, stability and quality control events. Reducing Cbp80 mRNA levels in the female germline revealed that Cbp80 is also involved in defending the germline against transposable elements. Combining such knockdown experiments with large scale sequencing of small RNAs further showed that Cbp80 is involved in the initial biogenesis of piRNAs as well as in the secondary biogenesis pathway, the ping-pong amplification cycle. We further found that Cbp80 knockdown not only led to the upregulation of transposons, but also to delocalization of Piwi, Aub and Ago3, key factors in the piRNA biosynthesis pathway. Furthermore, compared to controls, levels of Piwi and Aub were also reduced upon knock down of Cbp80. On the other hand, with the same treatment we could not detect significant changes in levels or subcellular distribution (nuage localization) of piRNA precursor transcripts. This shows that Cbp80 plays an important role in the production and localization of the protein components of the piRNA pathway and it seems to be less important for the production and export of the piRNA precursor transcripts.

Fig 1. Cbp80 phenotypes in the female germline and its role in silencing transposable elements.

(A) Germline-specific knock down of Cbp80 was performed either with the driver pCog-GAL4 and an shRNA against Cbp80 (shCbp80) or by the GAL4-nos.NGT40 driver combined with UAS-Dcr2 and a dsRNA against Cbp80 (dsCbp80). The knockdown leads to 3 phenotypic classes with the frequencies shown in (A’): normal wild-type phenotype [n], ovaries that appear to lack germ cells [gc^-], and partially developing ovaries that mostly degenerate after stages 7–9 [d]. (B-B’) Ovaries showing the "d" phenotype upon Cbp80 knockdown were tested for Cbp80 levels by Western blotting. The sample contained only a minor fraction of the other phentypes. In (B) wild-type ovaries and wild-type ovaries expressing a Myc-tagged Cbp80 were used as controls, and knock down was performed with the shRNA. 2 different amounts of the shCbp80 samples were loaded. In (B’) ovaries expressing a dsRNA against Cbp80 or GFP were used. Tubulin was used as a loading control. (C) Fold increases in RNA levels of indicated TEs upon germline-specific RNAi-mediated knock down of piwi and Cbp80 (shRNA against Cbp80). The germline GAL4 driver alone was used as control. Fold-changes in RNA levels relative to control were normalized to rp49 levels. Error bars indicate SD; n = 3, with 2 biological replicates. (D) Fold increases in RNA levels relative to rp49 of the same TEs upon knock down of Cbp80 using the dsRNA. Control ovaries expressed the dsGFP RNAi construct. The upregulation of the TE was also observed when Tub and BicD were used to normalize the reads (S3 Fig). *p<0.05; **p<0.01; ***p<0.001.

Fig 2. Germline Cbp80 is involved in producing piRNAs.

(A-C) Genotypes of ovaries analyzed are depicted on top. Ovaries showing the "d" phenotype (Fig 1A) upon Cbp80 knockdown were used. (A-C, F) Scale and axis depicted in the left pannel also apply to the corresponding panels on the right. (A-B) Histogram showing small RNAs (23–29 nucleotides) mapping to the soma-specific flamenco (flam) cluster (A) and to the germline-specific cluster 42AB (B). Ovaries expressed specifically in the germline shRNAs against Cbp80 or mCherry (as control). (C) Plots showing the size distribution of small RNAs derived from each strand of the 42AB and the flam clusters. Small RNA reads derived form the 42AB cluster were normalized to small RNAs mapping to the somatic flam, which is unaffected in this germline-specific knockdown. Number of small RNA reads of the characteristic size for piRNAs (23–29 nt) mapping to the germline-specific, dual-strand 42AB cluster were reduced upon Cbp80 knockdown. The same results were obtained when normalizing to the total number of small RNA reads. Small RNA reads derived form the flam cluster were normalized to the total number of small RNA reads. (D) A histogram showing the relative levels of 42AB derived piRNAs upon Cbp80 knockdown compared to the control knockdown. The data is normalized to the number of reads from the flam locus. Differences between control (shmCherry) and shCbp80 treatment are highly significant (p-value is < 2x10^-16 using a chi-square test). (E) Relative abundance of sense-antisense piRNA pairs overlapping by 10 nt (compared to the total number of sense-antisense pairs mapping to the flam or the 42AB clusters, in the small RNA libraries of the Cbp80 and the control knockdowns. Differences between 42AB control (shmCherry) and 42AB levels upon shCbp80 treatment are highly significant (p-value is < 2x10^-16 using a chi-square test). (F) Histograms showing the relative enrichment of RNAs overlapping by the indicated number of nucleotides, plotted by Z-score, for the 42AB and flam clusters. Knockdown targets in the female germline are indicated on top of the figure. The peak at position 10 (arrow) is indicative of a ping-pong signature.

Fig 3. Cbp80 is predominantly nuclear.

(A) Cbp80 signal (green) was revealed in wild-type ovaries using anti-Cbp80 antibodies. Ovaries expressing a Myc-tagged Cbp80 showed a similar fluorescence signal upon staining with an anti-Myc-epitope antibody. A similar pattern was also observed analyzing the Venus signal directly in ovaries expressing a Venus fusion protein. DNA staining is shown in blue. Anti-Aub staining (red) produces highest signal intensity in the perinuclear nuage with some additional cytoplasmic signal. (B) Co-staining for the nuclear envelope protein Lamin and tagged and untagged Cbp80 revealed that the Cbp80 signal is mainly inside the nuclei. (C) Cbp80 shows a very similar nuclear distribution pattern as Piwi. Right panels show high magnification of 2 different size nurse cell nuclei.

Fig 4. Cbp80 is needed for nuclear localization of Piwi.

(A) Germ line knockdown of ovarian Cbp80, targetting 2 different parts of Cbp80, and control knockdown (shmCherry). Wild-type egg chambers were an aditional control for quantification. Ovaries showing the "d" phenotype (Fig 1A) upon Cbp80 knockdown were dissected and co-stained with anti-Cbp80 antibodies (green signal) and anti-Piwi antibodies (red signal). DNA staining (Hoechst) is in blue. Egg chambers with reduced Cbp80 signal in the germline, but normal levels of nuclear Cbp80 signal in the surrounding follicule cells display a healthy normal appearance, but show mis-localization of Piwi protein to the cytoplasm. While rare cases of degenerating or damaged wild-type egg chambers can also show delocalization of Piwi in nurse cells and follicle cells, we did not observe this in healthy wild-type egg chambers. (B) Quantification of nuclear Piwi in the germline where Cbp80 expression is normal (Cbp80+) or efficiently knocked down (Cbp80-, where no or only very weak Cbp80 signal is seen in the germline). Scale bars: 25 μm in all ovariole pictures and 10 μm in magnified egg chamber pictures.

Fig 5. Cbp80 is needed for enrichment of Aub and Ago3 in the nuage.

Germline specific knockdown of Cbp80 and mCherry (control). Cbp80 knockdown ovaries of the "d" phenotype were used (Fig 1A). Egg chambers were co-stained with anti-Cbp80 antibodies (green signal) and anti-Aub (A, red signal), and anti-Ago3 (C, red signal), respectively. DNA staining (Hoechst) is in blue. (A) The localization of Aub (red), to the nuage is affected by knocking down Cbp80 with two different RNAi lines (shCbp80 and dsCbp80). The level of Cbp80 knockdown can be judged by comparing the Cbp80 signal in the huge nurse cell nuclei to the signal in the surrounding somatic follicel cell nuclei of the same egg chamber (internal control). This ratio is lower in the knock-down situation than in the controls. Egg chambers with Cbp80 knockdown show no clear nuage ring of Aub staining. Plots on the right of the pictures display the fluorescence signal intensity for each channel across the nurse cell nucleus shown. Scale bars: 7.5 μm in control and 10 μm in Cbp80 knockdown egg chamber pictures. (B) Quantification of perinuclear Aub accumulation in the germline where Cbp80 expression is normal or only slightly knocked down (Cbp80+), or where it is efficiently knocked down (Cbp80-, where no or only very weak Cbp80 signal is seen in the germline). The presence of the Aub ring in the nuage correlates with the presence of Cbp80. (C) In control egg chambers Ago3 (red) signal is slightly concentrated in the immediate vicinity of the nuclear envelope of the nurse cells, the nuage. Upon knock down of Cbp80, this localization is reduced or lost. Cbp80 staining (green) reveals efficiency of Cbp80 knockdown. Scale bars: 7.5μm. (D) Quantification of the correlation between Cbp80 levels and Ago3 localization to the nuage as done in B) for Aub localization.

Fig 6. Expression of piRNA pathway components requires Cbp80.

(A, D) Knock down of Cbp80 in egg chambers expressing a dsRNA against Cbp80 (dsCbp80) driven by the GAL4-nos.NGT40 driver and the UAS-Dcr2. Control flies expressed a dsRNA against GFP (dsGFP) (B, C) Ovaries expressing specifically in the germline (pCog-Gal4 driver) shRNAs against Cbp80 or mCherry (as control). Control flies used for RT-PCR experiments expressed also a Jupiter-mCherry fusion protein. (A-B) mRNA levels of piRNA pathway factors (piwi, aub, Rhi, zuc and Ago3) and Cbp80 were measured by qRT-PCR. Fold expression levels of each mRNA in the knockdown samples relative to its expression in the corresponding control are shown for each mRNA. Total starting RNA amounts were the same in both samples. Error bars represent ⁺/- SD of 2 controls in B and 3 control samples in A and 3 biological knockdown replicates. *p<0.05; **p<0.01; ***p<0.001. (C-D) Levels of Cbp80, Piwi, Aub, BicD, Cdk7, Clc and Tub (as loading control) were assessed by Western blotting. Ponceau staining is also shown to reveal total proteins as loading control. Levels Piwi, Aub and Cdk7 were strongly reduced upon Cbp80 reduction. On the other hand, levels of Tub, Clc and BicD were less affected. For Cbp80 knockdown samples were extracted from egg chambers showing the phenotype “d” (Fig 1A).