Ubiquitinome Profiling Reveals in Vivo UBE2D3 Targets and Implicates UBE2D3 in Protein Quality Control.

Ubiquitination has crucial roles in many cellular processes, and dysregulation of ubiquitin machinery enzymes can result in various forms of pathogenesis. Cells only have a limited set of ubiquitin-conjugating (E2) enzymes to support the ubiquitination of many cellular targets. As individual E2 enzymes have many different substrates and interactions between E2 enzymes and their substrates can be transient, it is challenging to define all in vivo substrates of an individual E2 and the cellular processes it affects. Particularly challenging in this respect is UBE2D3, an E2 enzyme with promiscuous activity in vitro but less defined roles in vivo. Here, we set out to identify in vivo targets of UBE2D3 by using stable isotope labeling by amino acids in cell culture-based and label-free quantitative ubiquitin diGly proteomics to study global proteome and ubiquitinome changes associated with UBE2D3 depletion. UBE2D3 depletion changed the global proteome, with the levels of proteins from metabolic pathways, in particular retinol metabolism, being the most affected. However, the impact of UBE2D3 depletion on the ubiquitinome was much more prominent. Interestingly, molecular pathways related to mRNA translation were the most affected. Indeed, we find that ubiquitination of the ribosomal proteins RPS10 and RPS20, critical for ribosome-associated protein quality control, is dependent on UBE2D3. We show by Targets of Ubiquitin Ligases Identified by Proteomics 2 methodology that RPS10 and RPS20 are direct targets of UBE2D3 and demonstrate that the catalytic activity of UBE2D3 is required to ubiquitinate RPS10 in vivo. In addition, our data suggest that UBE2D3 acts at multiple levels in autophagic protein quality control. Collectively, our findings show that depletion of an E2 enzyme in combination with quantitative diGly-based ubiquitinome profiling is a powerful tool to identify new in vivo E2 substrates, as we have done here for UBE2D3. Our work provides an important resource for further studies on the in vivo functions of UBE2D3.

Targeted Locus Amplification and Haplotyping.

Targeted locus amplification (TLA) allows for the detection of all genetic variation (including structural variation) in a genomic region of interest. As TLA is based on proximity ligation, variants can be linked to each other, thereby enabling allelic phasing and the generation of haplotypes. This allows for the study of genetic variants in an allele-specific manner. Here, we provide a step-by-step protocol for TLA sample preparation and a complete bioinformatics pipeline for the allelic phasing of TLA data. Additionally, to illustrate the protocol, we show the ability of TLA to re-sequence and haplotype the complete cystic fibrosis transmembrane (CFTR) gene (> 200 kb in size) from patient-derived intestinal organoids.

REV7: Jack of many trades.

The HORMA domain protein REV7, also known as MAD2L2, interacts with a variety of proteins and thereby contributes to the establishment of different complexes. With doing so, REV7 impacts a diverse range of cellular processes and gained increasing interest as more of its activities became uncovered. REV7 has important roles in translesion synthesis and mitotic progression, and acts as a central component in the recently discovered shieldin complex that operates in DNA double-strand break repair. Here we discuss the roles of REV7 in its various complexes, focusing on its activity in genome integrity maintenance. Moreover, we will describe current insights on REV7 structural features that allow it to be such a versatile protein.

MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5' end resection.

Appropriate repair of DNA lesions and the inhibition of DNA repair activities at telomeres are crucial to prevent genomic instability. By fuelling the generation of genetic alterations and by compromising cell viability, genomic instability is a driving force in cancer and ageing. Here we identify MAD2L2 (also known as MAD2B or REV7) through functional genetic screening as a novel factor controlling DNA repair activities at mammalian telomeres. We show that MAD2L2 accumulates at uncapped telomeres and promotes non-homologous end-joining (NHEJ)-mediated fusion of deprotected chromosome ends and genomic instability. MAD2L2 depletion causes elongated 3' telomeric overhangs, indicating that MAD2L2 inhibits 5' end resection. End resection blocks NHEJ while committing to homology-directed repair, and is under the control of 53BP1, RIF1 and PTIP. Consistent with MAD2L2 promoting NHEJ-mediated telomere fusion by inhibiting 5' end resection, knockdown of the nucleases CTIP or EXO1 partially restores telomere-driven genomic instability in MAD2L2-depleted cells. Control of DNA repair by MAD2L2 is not limited to telomeres. MAD2L2 also accumulates and inhibits end resection at irradiation-induced DNA double-strand breaks and promotes end-joining of DNA double-strand breaks in several settings, including during immunoglobulin class switch recombination. These activities of MAD2L2 depend on ATM kinase activity, RNF8, RNF168, 53BP1 and RIF1, but not on PTIP, REV1 and REV3, the latter two acting with MAD2L2 in translesion synthesis. Together, our data establish MAD2L2 as a crucial contributor to the control of DNA repair activity by 53BP1 that promotes NHEJ by inhibiting 5' end resection downstream of RIF1.