Abstract
The N-end rule pathway is a proteolytic system in which its recognition components (N-recognins) recognize destabilizing N-terminal residues of short-lived proteins as an essential element of specific degrons, called N-degrons. The RING E3 ligases UBR2 and UBR1 are major N-recognins that share size (200 kDa), conserved domains and substrate specificities to N-degrons. Despite the known function of the N-end rule pathway in degradation of cytosolic proteins, the major phenotype of UBR2-deficient male mice is infertility caused by arrest of spermatocytes at meiotic prophase I. UBR2-deficient spermatocytes are impaired in transcriptional silencing of sex chromosome-linked genes and ubiquitylation of histone H2A. In this study we show that the recruitment of UBR2 to meiotic chromosomes spatiotemporally correlates to the induction of chromatin-associated ubiquitylation, which is significantly impaired in UBR2-deficient spermatocytes. UBR2 functions as a scaffold E3 that promotes HR6B/UbcH2-dependent ubiquitylation of H2A and H2B but not H3 and H4, through a mechanism distinct from typical polyubiquitylation. The E3 activity of UBR2 in histone ubiquitylation is allosterically activated by dipeptides bearing destabilizing N-terminal residues. Insufficient monoubiquitylation and polyubiquitylation on UBR2-deficient meiotic chromosomes correlate to defects in double strand break (DSB) repair and other meiotic processes, resulting in pachytene arrest at stage IV and apoptosis. Some of these functions of UBR2 are observed in somatic cells, in which UBR2 is a chromatin-binding protein involved in chromatin-associated ubiquitylation upon DNA damage. UBR2-deficient somatic cells show an array of chromosomal abnormalities, including hyperproliferation, chromosome instability, and hypersensitivity to DNA damage-inducing reagents. UBR2-deficient mice enriched in C57 background die upon birth with defects in lung expansion and neural development. Thus, UBR2, known as the recognition component of a major cellular proteolytic system, is associated with chromatin and controls chromatin dynamics and gene expression in both germ cells and somatic cells.
Highlights
The N-end rule pathway is a ubiquitin (Ub)-dependent proteolytic system in which its recognition components (Nrecognins) recognize destabilizing N-terminal residues of shortlived proteins as an essential element of degradation signals, called N-degrons [1,2,3,4,5,6]
To characterize spatiotemporal distributions of Ub conjugates associated with meiotic chromatin, we stained surface-spread meiotic chromosomes from spermatocytes (n = 462, 5 mice) of P16 C57/129 mice using FK2 antibody known to detect both monoubiquitin and polyubiquitin conjugates [39]
UBR2 plays a genome-wide role in chromatin-associated ubiquitylation during meiotic prophase I in spermatocytes Given that more than 500 E3s function in virtually all known processes, we initially anticipated that meiotic chromosomes would be ubiquitously associated with a high level of Ub conjugates which represents various histone species and other chromatinassociated proteins
Summary
The N-end rule pathway is a ubiquitin (Ub)-dependent proteolytic system in which its recognition components (Nrecognins) recognize destabilizing N-terminal residues of shortlived proteins as an essential element of degradation signals, called N-degrons [1,2,3,4,5,6]. The ubiquitylation in the mammalian N-end rule pathway is mainly mediated by UBR1 and UBR2, two functionally overlapping RING finger E3 ligases with similarity in size (200 kDa), conserved domains, and specificities to N-degrons [6,9,10,16]. Known mammalian N-recognins share a 70-residue zinc-finger domain, called the UBR box, which binds type-1 N-terminal residues with a dissociation constant (Kd) of 1.6–3.4 mM [16,17,18,19]. The components, functions, and substrates of the N-end rule pathway are reviewed [2,3,5]
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