Early developmental arrest and impaired gastrointestinal homeostasis in U12-dependent splicing-defective Rnpc3-deficient mice

Abstract

Splicing is an essential step in eukaryotic gene expression. While the majority of introns is excised by the U2-dependent, or major class, spliceosome, the appropriate expression of a very small subset of genes depends on U12-dependent, or minor class, splicing. The U11/U12 65K protein (hereafter 65K), encoded by RNPC3, is one of seven proteins that are unique to the U12-dependent spliceosome, and previous studies including our own have established that it plays a role in plant and vertebrate development. To pinpoint the impact of 65K loss during mammalian development and in adulthood, we generated germline and conditional Rnpc3-deficient mice. Homozygous Rnpc3 −/− embryos died prior to blastocyst implantation, whereas Rnpc3 +/− mice were born at the expected frequency, achieved sexual maturity, and exhibited a completely normal lifespan. Systemic recombination of conditional Rnpc3 alleles in adult (Rnpc3 lox/lox ) mice caused rapid weight loss, leukopenia, and degeneration of the epithelial lining of the entire gastrointestinal tract, the latter due to increased cell death and a reduction in cell proliferation. Accompanying this, we observed a loss of both 65K and the pro-proliferative phospho-ERK1/2 proteins from the stem/progenitor cells at the base of intestinal crypts. RT-PCR analysis of RNA extracted from purified preparations of intestinal epithelial cells with recombined Rnpc3 lox alleles revealed increased frequency of U12-type intron retention in all transcripts tested. Our study, using a novel conditional mouse model of Rnpc3 deficiency, establishes that U12-dependent splicing is not only important during development but is indispensable throughout life.

Minor spliceosome disruption causes limb growth defects without altering patterning

“…In metazoans, the minor spliceosome, MIGs, and the position of minor introns within MIGs are all highly conserved 4–6 . This conservation could be explained by the enrichment of MIGs in essential functions required for cell survival 7 , which is consistent with embryonic lethality observed in multiple animal models with constitutive loss of minor spliceosome function 8–10 . Consequently, it is unsurprising that human diseases with minor spliceosome loss-of-function mutations have not been discovered.…”

Minor Splicing Factors Zrsr1 and Zrsr2 Are Essential for Early Embryo Development and 2-Cell-Like Conversion

“…These authors indicate that embryos fail to develop beyond the morula stage, similar to our results obtained in Zrsr1/2 mu mice, where only a few embryos continue developing via abnormal divisions after the 2-cell stage until the morula stage. In the study by Doggett et al [20], the mother was heterozygous for the Rnpc3 mutation, and thus the embryo could undergo the first few divisions using the protein product of the WT maternal allele in the oocyte, reaching a further developmental stage than in our experiment. We also showed the complementary behavior of Zrsr1 and Zrsr2 during preimplantation development.…”

“…According to a recent study, minor splicing is essential for preimplantation development [20]. Knockout of Rnpc3, coding for one of seven proteins unique to the U12-dependent spliceosome carrying a U12 intron, blocks preimplantation development in mice [20].…”

“…According to a recent study, minor splicing is essential for preimplantation development [20]. Knockout of Rnpc3, coding for one of seven proteins unique to the U12-dependent spliceosome carrying a U12 intron, blocks preimplantation development in mice [20]. These authors indicate that embryos fail to develop beyond the morula stage, similar to our results obtained in Zrsr1/2 mu mice, where only a few embryos continue developing via abnormal divisions after the 2-cell stage until the morula stage.…”

“…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]. Additionally, the minor spliceosome is thought to play an important developmental role in plants [12][13][14], drosophila [15] zebrafish [16], and in the mouse central nervous system [17,18], hypothalamus [19], gametogenesis [9], and early development [20]. Systematic transcriptome and proteome analyses of mouse and human preimplantation embryos have revealed that genes involved in mRNA splicing are over-represented during early preimplantation development [21], before and during zygotic gene activation (ZGA) [22].…”