Investigating the function of E3 ubiquitin ligase, UBR2, in chromosome stability

Abstract

Ubiquitination is an essential cellular mechanism where ubiquitin attaches to proteins to regulate their function or target them for degradation via the proteasome. The transfer of ubiquitin from E2 ubiquitin conjugating enzymes to target proteins is regulated by the highly diverse group of E3 ubiquitin ligases. One prominent family of the E3 ubiquitin ligases are the seven UBR proteins, four of which have overlapping roles in the N-end rule pathway where they, canonically, bind either basic (Type I) or bulky hydrophobic (Type II) amino acids at the N-terminus of the substrate. However, at least some of these UBR proteins also have non-canonical roles outwith the N-end rule pathway, and even non-catalytic roles in stabilising some proteins. Null genetic mutations in Ubr2 cause increased amounts of cohesin associating with chromosomes, defects in chromosome synapsis during meiosis, male infertility and female-specific embryonic lethality. However, it is not clear whether these phenotypes relate to UBR2’s role in the N-end rule pathway or its non-canonical roles.

As UBR2 has a role in chromatin-bound cohesin maintenance in mitotic cells, I investigated whether UBR2 might localise to chromosomes to regulate chromatin-associated cohesin directly. I overexpressed FLAG-tagged UBR2 in HEK293T cells and imaged the cells throughout the cell cycle. Although I could not detect localisation of wild-type UBR2 to chromatin at any stage of the cell cycle, a version of UBR2 with mutations in its ELL domain did localise to chromosomes during mitosis in 30% of HEK293T cells suggesting that this mutation increases the affinity of UBR2 to bind to chromosomes and regulate chromosome-associated substrates.

Next, I wanted to investigate how UBR2 regulates chromosome-associated substrates. In order to dissect which domains of UBR2 are involved in this function, I used CRISPR-Cas9 technology to introduce separation-of-function point mutations into the endogenous Ubr2 locus, and characterised the effects of these mutations on known UBR2 substrates. Using these cells along with Ubr2-/- cells, I was able to use mass spectrometry to identify potential N-end rule substrates and non-canonical substrates of UBR2.

Finally, I used CRISPR-Cas9 technology to introduce these separation-of-function mutations into mice in order to determine how the distinct biochemical roles of UBR2 relate to the Ubr2-/- null mouse phenotypes. Surprisingly, I found that the major phenotypes in Ubr2-/- mice are not recapitulated by mutations that specifically affect N-end rule substrate ubiquitination, that non-canonical protein ubiquitination is likely involved in Ubr2-dependent regulation of cohesin, and that the well-established role for UBR2 during spermatogenesis does not appear to act through either N-end rule or non-canonical UBR2-dependent ubiquitination. I propose that the key physiological role of UBR2 in meiosis and spermatogenesis relies on non-catalytic roles of UBR2 in binding to, and stabilising, its binding partner TEX19.1.