ncRNA BC1 influences translation in the oocyte

Figures & data

Figure 1. BC1 ncRNA is abundant in the GV oocyte with polysomal association

(A) qRT-PCR analysis of BC1 expression in the GV, MII oocytes and 2-cell embryo. Values obtained for GV stage were set as 1. Gapdh mRNA and Neat2 lncRNA were used as controls. Relative expression; mean ± SD; Student’s t-test: **p< 0.01; ***p< 0.001, ns for non-significant, n=3. (B) Expression of BC1 in the nucleus and cytoplasm of oocyte. Values obtained from qPCR of the nucleus and cytoplasm combined as 100%. Gapdh mRNA and Neat2 lncRNA were used as controls. See also Figure S1. (C) Polysomal association of RNAs coding for Gapdh, Neat2 and BC1 in the GV and MII oocyte. Expression in the GV stage was set as 1. Gapdh mRNA and Neat2 lncRNA were used as controls. Relative expression, mean ± SD; Student’s t-test: *p< 0.05; ***p< 0.001, ns for non-significant, n ≥ 2. NP, non-polysomal; P, polysomal. See Figure S2 for validation of polysomal fractionation.

Figure 2. BC1 overexpression leads to decrease of global translation and TOP RNA reporter in the GV oocytes

(A) ³⁵S-Methionine incorporation in mouse oocytes with overexpression (OE) of BC1 RNA. Expression GAPDH protein was used as a loading control. Representative images from at least three independent replicates. See Figure S3 for validation of overexpression. (B) Quantification of ³⁵S-Methionine incorporation. Values obtained for the control (Cntr) group were set as 1. Data from four independent experiments; mean ± SD; Student’s t-test: *p<0.05; ns, non-significant, n≥4. (C) Analysis of NanoLuc expression of RNA reporters in oocytes contains mutation in 5ʹUTR (mut) and with canonical 5ʹTOP motive in the control and OE of BC1 RNA. Chemiluminescence values obtained for the Cntr group were set as 1 and Firefly Chemiluminescence was used as a microinjection control for normalization (Nluc/FL); mean ± SD; Student’s t-test: **p< 0.01; ns, non-significant, n ≥5.

Figure 3. BC1 ncRNA influences translation of specific targets in the GV mouse oocyte

(A) Western blot analysis of ACTB, DLG4 and SVV in GV and MII oocytes with OE of BC1RNA. GAPDH was used as a loading control. Representative images from at least three independent replicates. (B) Quantification of western blot analysis. Values obtained for the control groups injected with H2b:gfp RNA were set as 1. Data from three independent experiments; mean ± SD; Student’s t-test: *p<0.05; ***p<0.001; ns, non-significant.

Figure 4. BC1 physically interacts with candidate mRNAs via protein interplay

(A) Scheme of BC1 anti-sense DNA 5ʹ-biotinylated oligonucleotide probes related to BC1. (B) Pull-down from whole oocytes lysate with three probes complementary to BC1 ncRNA (BC1) and without probes as a negative control (Cntr). Endogenous BC1 and Gapdh RNAs were used as additional controls. Values obtained from control were set as 1. Relative expression; mean ± SD; Student’s t-test: *p< 0.05; **p< 0.01, ***p< 0.001, ns for non-significant, n≥2. (C) Pull-down from purified total RNA with three probes complementary to BC1 ncRNA (BC1) and without probes as a negative control (Cntr). Endogenous BC1 and Gapdh RNAs were used as additional controls. Values obtained from control were set as 1. Relative expression; mean ± SD; Student’s t-test: *p< 0.05; ns for non-significant, n=3.

Figure 5. BC1 interacts with FMRP protein in the GV oocyte

(A) RNA-protein proximity ligation assay (RNA-PLA) detecting BC1 ncRNA–FMRP interactions. Representative confocal images of GV and MII oocytes; scale bars 25 µm; PLA green; DNA blue; n ≥ 7. (B) Quantification of BC1–FMRP interactions in GV and MII oocytes. Values obtained for the GV oocytes were set as 1. Relative number of BC1–FMRP interactions; mean ± SD; Student’s t-test: ***p<0.001, n≥7.

Figure 6. BC1 overexpression does not lead to clustering of FMRP and RNA in GV oocyte

(A) Representative confocal images of GV and MII oocytes labelled with FMRP antibodies in the absence (Cntr) or presence (BC1 OE) of BC1; scale bars 25 µm; FMRP red; DNA blue; n ≥ 30. (B) Quantification of FMRP clusters in MII oocyte. Values obtained for the Cntr group were set as 1. Relative number of FMRP clusters; mean ± SD; Student’s t-test: ***p<0.001, n≥30. (C) Representative confocal images of GV and MII oocytes labelled with Oligo(dT) Probe for poly(A) RNA in the absence (Cntr) or presence (BC1 OE) of BC1; scale bars 25 µm; FMRP red; DNA blue; n ≥ 10. (D) Quantification of poly(A) RNA clusters in MII oocyte. Values obtained for the Cntr groups were set as 1. Relative number of poly(A) RNA clusters; mean ± SD; Student’s t-test: **p<0.01, n≥10.

Figure 7. Overexpression of BC1 does not alter oocyte or early embryo development

(A) Analysis of the maturation and developmental competence of the oocyte with BC1 overexpression. Relative number of oocytes; mean ± SD; Student’s t-test: ns for non-significant, n ≥ 42. See Figure S3 for validation of overexpression. (B) Analysis of the morphology of the MII oocyte with overexpression of BC1. Representative confocal images of MII oocytes labelled with tubulin antibodies in the control (Cntr) and OE of BC1; scale bars 25 µm; Tubulin green; DNA blue; n ≥ 20. (C) Bright-field images of blastocyst development in control and BC1 OE groups.