Fig 2: H3K36me3 is regulated through the Pi3k/Akt pathway. (A) Treatment with a pan Akt inhibitor (MK-2206) or a Pten inhibitor (VO-OHpic) blocks mouse embryo development at the 2-cell stage, whereas treatment with an Akt2 inhibitor (CCT128930) does not affect blastocyst formation. Scale bar: 50 µm. The graph depicts the development rates of treated embryos. (B) Inhibition of pan Akt dramatically decreases the global level of H3K36me3 but not that of H3 in mouse 2-cell embryos. Inhibition of Pten significantly increases the level of H3K36me3 but not that of H3. (C) Inhibition of pan Akt or Pi3K by LY-294002, but not Akt2, reduces the levels of both H3K36me3 and Pol II CTD Ser2p, while inhibition of Pten or inhibition of Kdm4 by ML-324 increases the levels of both modifications. Concurrent inhibition of pan Akt and Pten does not significantly affect H3K36me3 or Pol II CTD Ser2p. The graph depicts the mean fluorescence intensities of both modifications in 2-cell embryos after the indicated treatments. In total, 234 embryos were used for this experiment. (D) Inhibition of Akt does not significantly affect the nuclear levels of pan Pol II or Setd2. (E) PLA shows that pan Akt inhibition disrupts the interaction of Setd2 with H3K36me2. The fluorescence signal is very low in MK-2206-treated embryos compared to their DMSO-treated counterparts. (F) Double-immunostaining shows that pan Akt colocalizes with Iws1 in the cell nuclei of 2-cell embryos. Akt inhibition reduces the nuclear signal of Akt but not that of Iws1. PLA reveals that Akt kinase activity is required for the Akt-Iws1 interaction. (G) Akt kinase activity is required for proper mRNA nuclear export. MK-2206 induces bulk poly(A)+ accumulation in 2-cell embryos. The fluorescence intensity of nuclear poly(A)+ is significantly higher in MK-2206-treated 2-cell embryos compared to DMSO-treated 2-cell embryos. (H) PLA shows that Akt kinase activity is required for the proper interaction of Alyref with Pol II CTD Ser2p (CTD S2p). The fluorescence intensity of the PLA nuclear signal is significantly lower in MK-2206-treated 2-cell embryos compared to their DMSO-treated counterparts. DNA was counterstained with DAPI. At least 20 embryos were used for each experiment. All experiments were repeated at least three times. Scale bars: 20 µm.

Fig 3: Effects of siIws1 or siSupt6 on mRNA splicing and export in mouse embryos. (A) Zygotes were electroporated with siControl, siIws1 or siSupt6 and cultured to the blastocyst stage. At the indicated times, splicing speckles were detected using anti-SC35. At least 15 embryos were used for each group. The fluorescence intensity of SC35 was measured in each specimen; mean values are depicted in the graph. (B) The pre-mRNA splicings of Cdk9 and c-Myc are defective in siIws1 and siSupt6 embryos. The levels of spliced and unspliced intron 1 were quantified by RT-qPCR. The expression level of the 18 S rRNA in siControl was set as 1 and used as an internal control. (C) Bulk Poly(A)+ mRNA accumulation in siIws1 or siSupt6 embryos. Poly (A)+ mRNAs were labeled by FISH using a specific oligo-dT probe and analyzed by confocal microscopy. The fluorescence intensities are depicted in the graph. At least 15 embryos were analyzed for each stage. (D) Iws1 colocalizes and interacts with Alyref. Four-cell embryos were double-immunostained against Iws1 and Alyref. PLA shows the putative interaction of the two factors. DNA was counterstained with DAPI. (E) Four-cell stage siControl, siIws1, and siSupt6 embryos were double-stained for Alyref and Pol II CTD Ser2p. PLA shows that either Iws1 or Supr6 is required for the interaction of Alyref with Pol II CTD Ser2p. Scale bars: 20 µm.

Fig 4: ZC3H14 interacts with the THO complex. (A) Co-immunoprecipitation of ZC3H14 and THO components from mouse brain lysate. The Input and immunoprecipitated samples for ZC3H14, ZC3H14 + RNase, and control IgG are shown. THOC1 and THOC2 serve as representative members of the THO complex, together with the THO-associated protein ALYREF. HuR serves as a negative control. Asterisks indicate non-specific protein bands detected by the commercial antibodies. (B) Reciprocal co-immunoprecipitation using anti-THOC1. The Input and immunoprecipitated samples for THOC1, THOC1 + RNase and control IgG immunoprecipitations were probed to detect THOC1, ZC3H14 and THOC2 proteins. (C) Sedimentation of ZC3H14 protein complexes from the nuclear fraction of mouse brain lysate through a 10–50% glycerol gradient. Gradient fractions from top to bottom were resolved on SDS-PAGE from left to right. Fractions were probed with antibodies to detect ZC3H14, THOC1, THOC2, ALYREF and Nucleophosmin (NPM1) as a control. The Input sample is shown on the left. Overlapping fractions are outlined. Molecular weights are shown in kDa and indicated to the left.

Fig 5: ZC3H14 and THO components bind to common mRNA targets. (A) Endogenous ZC3H14, THOC5 and ALYREF RNA immunoprecipitation from N2a cells. The Input fractions and the Bound immunoprecipitated fractions were compared to the IgG immunoprecipitated control to confirm efficient and specific immunoprecipitation of ZC3H14, THOC5, and ALYREF. Molecular weights are indicated in kDa to the left. Asterisk indicates a non-specific band recognized by the commercial ALYREF antibody. (B–E) Quantification of RNA IP enrichment in (A). RNA isolated from the RNA-IPs was subjected to RT-qPCR analyses to detect the (B) Atp5g1, (C) Psd95, (D) Mapt and (E) Rplp0 transcripts. RNA levels in the bound fractions are normalized to input levels and are presented as fold change normalized to IgG control, with the value of the IgG control set to 1.0. Values represent the mean ± SEM for n = 3 independent experiments. Asterisks (*) indicate results that are statistical significance at *P-value < 0.05.