Functional roles of hnRNPA2/B1 regulated by METTL3 in mammalian embryonic development

Abstract

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) plays an important role in RNA processing via in m⁶A modification of pre-mRNA or pre-miRNA. However, the functional role of and relationship between m⁶A and hnRNPA2/B1 in early embryonic development are unclear. Here, we found that hnRNPA2/B1 is crucial for early embryonic development by virtue of regulating specific gene transcripts. HnRNPA2/B1 was localized to the nucleus and cytoplasm during subsequent embryonic development, starting at fertilization. Knockdown of hnRNPA2/B1 delayed embryonic development after the 4-cell stage and blocked further development. RNA-Seq analysis revealed changes in the global expression patterns of genes involved in transcription, translation, cell cycle, embryonic stem cell differentiation, and RNA methylation in hnRNPA2/B1 KD blastocysts. The levels of the inner cell mass markers OCT4 and SOX2 were decreased in hnRNPA2/B1 KD blastocysts, whereas that of the differentiation marker GATA4 was decreased. N6-Adenosine methyltransferase METTL3 knock-down caused embryonic developmental defects similar to those in hnRNPA2/B1 KD embryos. Moreover, METTL3 KD blastocysts showed increased mis-localization of hnRNPA2/B1 and decreased m⁶A RNA methylation. Taken together, our results suggest that hnRNPA2/B1 is essential for early embryogenesis through the regulation of transcription-related factors and determination of cell fate transition. Moreover, hnRNPA2/B1 is regulated by METTL3-dependent m⁶A RNA methylation.

Figure 1

Developmental expression and localization of hnRNPA2/B1 in mouse preimplantation embryos. (A) Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of hnRNPA2/B1 transcript levels in 1-cell (1C), 2-cell (2C), 4-cell (4C), morula (Mo), and blastocyst (BL) stages. (C) Immunocytochemistry (ICC) analysis revealed the hnRNPA2/B1 protein in all the nuclei during preimplantation development. Error bars indicate the mean ± s.e.m. Scale bars = 50 μm.

Figure 2

Effects of RNAi-mediated knock-down of hnRNPA2/B1 on mouse preimplantation embryo development. (A) Immunocytochemistry (ICC) between the control and hnRNPA2/B1 KD groups. Each embryo was microinjected at the zygote stage and cultured for 24 h. hnRNPA2/B1 intensity (B) and mRNA levels (C) were confirmed. Developmental competence between negative (D) and hnRNPA2/B1 KD embryos (E) were observed by time-lapse microscopy at 24 h after injection of eGFP dsRNA and hnRNPA2/B1 dsRNA into zygotes. DNA was visualized by cRNA encoding H2B–mCherry (red). Time-lapse experiments were repeated three times for each group. (F–H) Time course developmental changes were examined at the 4C, morula, and blastocyst stages. Developmental stages were evaluated by chromatin status. Error bars indicate the mean ± s.d. ***P < 0.01. Scale bars = 50 μm.

Figure 3

hnRNPA2/B1 is required for the development of post-implantation embryos. (A,B) Representative images showing blastocyst and outgrowth analysis using the control and hnRNPA2/B1 knockdown blastocysts. Rate of ICM-derived colony formation in the control and hnRNPA2/B1 knockdown blastocyst after 48 and 144 h. (C) The mean blastocyst size was calculated at 96 h. (D) The rate of ICM-derived colony formation in the control and hnRNPA2/B1 knockdown blastocyst after seeding at 48 and 144 h. 30–40 embryos were used in each group. The dashed line represents the area of attached colonies. Error bars indicate the mean ± s.d. **P < 0.05, ***P < 0.01. Scale bars = 100 μm.

Figure 4

RNA-Seq analysis of the control and hnRNPA2/B1 KD blastocysts. (A) Heatmap comparing the transcription level of up-regulated genes or down-regulated genes at the blastocyst stage. The expression level of genes was significantly (FRKM > 2, or <2) increased or decreased compared with that of the control. Con: Control, KD: hnRNPA2/B1 KD. (B,C) Composition of up-regulated and down-regulated genes in hnRNAP2/B1 KD blastocyst at 96 h. A total of 5,846 genes was downregulated upon hnRNPA2/B1 KD, and over 45% of which are transcription-related genes. A total of 6,060 genes was up-regulated upon hnRNPA2/B1 KD, and over 50% of which are transcription-related genes.

Figure 5

Gene expression determined by quantitative real-time PCR analysis of various gene mRNAs in the control and hnRNPA2/B1 KD embryos at the blastocyst stage. Three experimental replicates were carried out. ***P < 0.01. Error bars indicate the mean ± s.e.m.

Figure 6

Effects of hnRNPA2/B1 knockdown on pluripotency-related gene expression. (A,B) OCT4 and CDX2 and (C,D) SOX2 and CDX2 double-immunostaining in the control and hnRNPA2/B1 KD blastocysts (red: OCT4, red: SOX2, green: CDX2, blue: DNA). 5–10 embryos were used in each experiment (n = 3). (E,F) GATA4 immunostaining in the control and hnRNPA2/B1 KD blastocyst (green; GATA, blue; DNA). The dashed line represents area of the ICM. Error bars indicate the mean ± s.e.m. ***P < 0.01. Scale bars = 50 μm.

Figure 7

Effect of METTL3 during the early embryo development. (A) mRNA expression levels analysed by qRT-PCR and (B) localization by ICC GV at the blastocyst stage. (C) ICC of METTL3 in the control and METTL3 KD 2-cell embryos. Each embryo was microinjected at the zygote stage and cultured for 24 h. (D) METTL3 intensity and (E) mRNA levels were examined to evaluate the efficiency of METTL3 KD. (F) Representative images showing blastocyst and outgrowth analysis using the control and METTL3 knockdown blastocysts. (G) Rate of blastocyst and (H) ICM-derived colony formation in control and METTL3 knockdown blastocysts after 144 h. Three experimental replicates were examined using 20–30 embryos per group. The dashed line represents the area of nuclei. ***P < 0.01. Error bars indicate the mean ± s.d.

Figure 8

Effects of RNAi-mediated knockdown of METTL3 on m⁶A RNA methylation and hnRNPA2/B1 localization. (A) ICC analysis of N6-methyladenosin (m⁶A) in the control and METTL3 KD 4-cell embryos. METTL3 localized in the cytoplasm and decreased m⁶A intensity by METTL3 KD embryos. (C,D) ICC of m⁶A images in the control, hnRNPA2/B1 KD, and METTL3 KD 4D blastocysts. (E,F) Immunocytochemistry detection of hnRNPA2/B1 in the control and METTL3 KD blastocysts. 30–40 embryos were used in each group. ***P < 0.01. Error bars indicate the mean ± s.d. Scale bars = 50 μm.