G-quadruplex (G4) is a unique nucleic acid secondary structure formed by guanine-rich DNA or RNA sequences through Hoogsteen hydrogen bonding among four guanines. DHX36 (also known as RHAU) has been identified as a G4 helicase captured and purified from human cell lysates. Studies have shown that DHX36 participates in intracellular transcription and post-transcriptional processes by unwinding G4 structures, playing a crucial role in physiological processes such as spermatogenesis and heart development. However, the function of DHX36 in mammalian oocytes and early embryonic development remains unclear.
Researchers found that DHX36 is highly expressed in mouse ovarian tissue and primarily localized in the nucleus. As oocytes develop, nucleolar conformation undergoes transformation, and DHX36 gradually relocates from the nucleoplasm to the nucleolus. To investigate the role of DHX36 in oocyte development, the research team constructed an oocyte-specific conditional knockout (CKO) mouse model using the Cre-LoxP system. They discovered that DHX36-CKO mice were completely infertile, accompanied by severe hormonal response defects and ovulation disorders.
Further studies revealed that meiotic maturation and post-fertilization embryonic development were impaired in DHX36-CKO oocytes, manifesting as follows:
(1) Most oocytes were arrested during meiosis, failing to undergo germinal vesicle breakdown (GVBD) or extrude the first polar body.
(2) In oocytes that extruded the first polar body, chromosome alignment, and spindle assembly were disrupted, with some oocytes failing to maintain normal ploidy.
(3) Most oocytes from natural mating failed to fertilize and form pronuclei.
(4) Fertilized eggs predominantly arrested at the zygote stage, with only a few progressing to the 2-cell stage before stalling.
To explore the mechanisms underlying DHX36's role in oocyte function, researchers analyzed chromatin conformation and openness. They found that nucleolus-like bodies (NLBs) were enlarged in DHX36-CKO oocytes, heterochromatin condensation was reduced, and histone modifications (H3K4me3, H3K9me3, and H3K27me3) were significantly diminished. The heterochromatin marker protein HP1α exhibited a more diffuse distribution. ATAC-Seq analysis showed that DHX36 deletion reduced chromatin openness, particularly at transcription start sites and rDNA open regions, which were enriched with G4 sequences.
Using Smart-seq2 technology, the team conducted RNA sequencing (RNA-seq) on oocytes at various stages and fertilized eggs. They observed insufficient transcription and impaired clearance of maternal transcripts. Gene Ontology analysis revealed that differentially expressed genes were primarily enriched in pathways related to DNA transcription factor activity, ribonucleoprotein biogenesis, and rRNA processing. Further investigations demonstrated dysregulation of pre-rRNA in DHX36-CKO oocytes, with levels insufficient in growing oocytes (GO) and abnormally accumulated in fully-grown oocytes (FGO), accompanied by significantly reduced translational activity.
Employing the G4 fluorescent probe (CYTO) and G4-specific antibody (BG4), the study revealed significant G4 accumulation in the nucleolus and nucleoplasm of DHX36-CKO oocytes. The rDNA coding region (identical to the pre-rRNA sequence) was rich in G4 sequences, which formed G4 structures in vitro and bound to DHX36. The researchers hypothesized that DHX36 deficiency leads to G4 accumulation on rDNA, inhibiting pre-rRNA transcription.
Why does pre-rRNA accumulate in DHX36-CKO FGO? In vitro experiments revealed that treating cells with the G4 stabilizer PDS made pre-rRNA more resistant to RNase digestion. The distribution of DHX36, pre-rRNA, and G4 structures in early embryos showed high spatial overlap. RIP-qPCR analysis demonstrated that DHX36 directly binds pre-rRNA through its specific domain (RSM). Reintroducing DHX36 alleviated the accumulation of pre-rRNA in CKO oocytes. The study concluded that DHX36 ensures proper pre-rRNA processing during the transition from GO to FGO by resolving G4 structures. In DHX36-CKO oocytes, the accumulation of G4 structures impedes pre-rRNA cleavage, ultimately disrupting ribosome assembly and translation.
