Abstract
Minor splicing plays an important role in vertebrate development. Zrsr1 and Zrsr2 paralog genes have essential roles in alternative splicing, mainly participating in the recognition of minor (U12) introns. To further explore their roles during early embryo development, we produced Zrsr1mu and Zrsr2mu mutant mice, containing truncating mutations within the second zinc finger domain. Both homozygous mutant mice were viable with a normal lifespan. When we crossed a homozygous Zrsr2mu/mu female with Zrsr1mu/mu male, the double heterozygotes were non-viable, giving rise to embryos that stopped developing mainly between the 2- and 4-cell stages, just after zygotic gene activation. RNA-seq analysis of Zrsr1/2mu 2-cell embryos showed altered gene and isoform expression of thousands of genes enriched in gene ontology terms and biological pathways related to ribosome, RNA transport, spliceosome, and essential zygotic gene activation steps. Alternative splicing was analyzed, showing a significant increase in intron retention in both U2 and U12 intron-containing genes related to cell cycle and mitotic nuclear division. Remarkably, both Zrsr1 and Zrsr2 were required for the conversion of mouse-induced pluripotent stem cells into 2C-like cells. According to our results, Zrsr1 or Zrsr2 are necessary for ZGA and both are indispensable for the conversion of induced pluripotent stem cells into 2C-like cells.
Highlights
Alternative splicing (AS) is an important co- and post-transcriptional process through which multiple transcripts are generated from a single gene
RNA-seq analysis of Zrsr1/2mu 2-cell embryos showed altered gene and isoform expression of thousands of genes enriched in gene ontology terms and biological pathways related to ribosome, RNA transport, spliceosome, and essential zygotic gene activation steps
Zrsr1 or Zrsr2 are necessary for zygotic gene activation (ZGA) and both are indispensable for the conversion of induced pluripotent stem cells into 2C-like cells
Summary
Alternative splicing (AS) is an important co- and post-transcriptional process through which multiple transcripts are generated from a single gene. Genes with U12 introns are over-represented in functions and pathways related to development, such as RNA processing, DNA replication, or cell cycle [4,5] Mutations within both the protein and snRNA components of the minor spliceosome have been associated with multiple diseases, including developmental disorders [4,6,7,8,9], neurodegeneration [10], and cancer [11]. Recent sequencing studies have identified frequent somatic ZRSR2 mutations in hematological malignancies, such as myelodysplastic syndrome (MDS), causing mis-splicing of U12 introns [11] Their precise role in embryo development and function in other pluripotent cell types remains unclear. This study identifies ZRSR1 and ZRSR2 as essential factors for efficient U2 and U12 intron splicing and reveals their crucial roles in genome activation during both ZGA and conversion of induced pluripotent stem cells (iPSC) into 2C-like cells (2CLC)
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