Uncovering non-canonical functions for the E3 ubiquitin ligase UBR2 in the mouse

Ubiquitination is an essential cellular mechanism in which the small polypeptide ubiquitin is attached to proteins, so regulating protein function or targeting them for degradation via the proteasome. The transfer of ubiquitin to specific target proteins is regulated by the highly diverse group of E3 ubiquitin ligases. These require two key domains: a substrate recognition site, and a ubiquitin carrier E2 protein binding site, which allow close proximity of the two, so ubiquitin transfer can occur. One family of E3 ubiquitin ligases are the UBR proteins, which confer substrate specificity through the N-end Rule pathway – in which they, canonically, bind either Type I (basic, e.g. Arginine) or Type II (bulky hydrophobic, e.g. Phenylalanine) amino acids on the N-terminus of the substrate. There are seven known UBR proteins, and UBR2 is one of four in this family that are thought to have overlapping roles in the N-end Rule pathway. Published murine phenotypes in a Ubr2-/- mouse include male infertility and female specific embryonic lethality. The tissue-specificity of the Ubr2-/- phenotype has been attributed to tissue-specific expression of UBR proteins allowing critical functions of the N end rule pathway to become detectable in specific cell types. These phenotypes also appear to overlap that of a Tex19.1-/- mouse, which has undetectable expression of the UBR2 interacting protein TEX19.1. This is specifically expressed in hypomethylated tissues: the testes, ovaries, placenta, and in a mouse embryonic stem cell model. In addition to the male infertility observed in both mouse models, Tex19.1-/- phenotypes include placental defects associated with intrauterine growth restriction, and cohesin misregulation during female meiosis, in the oocyte. There is some evidence that UBR2’s interaction with TEX19.1 regulates its Type II N-end Rule activity, and so I hypothesized that UBR2 also has roles in the other phenotypes of the Tex19.1-/- mouse, and that these act through the N-end Rule, which has thus far not been directly shown in any of the phenotypes present. Firstly, I aimed to study a potential role for UBR2 during female meiosis. In oocytes, cohesin molecules hold homologous chromosomes in a tightly aligned conformation. This must be maintained for long periods of time, between meiotic prophase in the embryonic ovary and resumption of meiosis prior to ovulation during adulthood. This maintains chromosome stability and prevents missegregation during the first meiotic division, leading to aneuploidy and reduced fertility. A longer arrest period, i.e. in oocytes isolated from older females, is associated with loss of cohesin and increased chance of aneuploidy. In absence of UBR2 in oocytes from young females, less cohesin is present on the chromosome arms at the first meiotic metaphase and homologs are less tightly held together, so implying a role for protection of chromosome cohesion over time. Next, in accordance with previous observations in absence of UBR2, I identified an embryonic lethality present in our Ubr2-/- mice, which is associated with intrauterine growth restriction and placental defects. Although this is superficially similar to the Tex19.1-/- placental phenotype, there also appear to be TEX19.1-independent functions of UBR2 in this tissue. Finally, I aimed to ascertain the mechanism by which UBR2 plays a role in any of these phenotypes. As UBR2 is thought to confer particular specificity towards Type II N-end Rule substrates, and TEX19.1 is thought to regulate this role of UBR2, I used CRISPR-Cas9 technology to generate an Ubr2T2/T2 mouse model, which has undetectable Type II substrate binding. There are no gross phenotypes in any of the tissues identified using the Ubr2-/- null mouse; both placental growth and meiotic cohesin appear to be normally regulated, and so these functions of UBR2 do not appear to be acting through the N-end Rule pathway. Additionally, the well-established role for UBR2 during spermatogenesis does not appear to act through Type II N-end Rule substrate binding, as has been assumed. Instead, I conclude that, although UBR2 is an important part of the N-end Rule pathway in other contexts, it has key non-canonical functions in both male and female fertility, and in placental development, which cannot be compensated for by other UBR proteins