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RNF212B E3 ligase is essential for crossover designation and maturation during male and female meiosis in the mouse

ABSTRACT: Meiosis, a reductional cell division, relies on precise initiation, maturation and resolution of crossovers (COs) during prophase I to ensure the accurate segregation of homologous chromosomes during metaphase I. This process is regulated by the interplay of RING-E3 ligases such as RNF212 and HEI10 in mammals. In this study, we characterized a novel RNF212B E3 ligase. RNF212B colocalizes and interacts with RNF212 forming small foci along meiotic chromosome cores from zygonema onwards. These consolidate into larger foci at maturing COs, colocalizing with HEI10, CNTD1, and MLH1 at pachynema in a synapsis-dependent and DSB-independent manner. Genetically, RNF212B focus formation depends on the presence of Rnf212 and Msh4 but not on Hei10 and Cntd1, while the unloading of RNF212B at the end of pachynema is dependent on Hei10 and Cntd1. Rnf212b-deficient and enzymatically-dead mutant mice exhibit modest synapsis defects, a reduction in localization of pro-CO factors (MSH4, TEX11, RPA, MZIP2) and absence of MLH1 foci, indicative of nascent COs, resulting in metaphase I cells with mostly univalent chromosomes. Double mutants for Rnf212b and Rnf212 exhibit an identical phenotype to that of Rnf212b single mutant males, while double heterozygous demonstrate a dosage-dependent reduction in the number of COs relative to the single mutant, indicating a functional interplay between both paralogs. SUMOylome analysis of testis from Rnf212b mutants and pull-down analysis of Sumo- and Ubiquitin-tagged HeLa cells, suggest that RNF212B is a novel E3 ligase with Ubiquitin activity, serving as a crucial factor for CO designation and maturation. This conserved pathway holds physiological significance for understanding the biology and homesotais of CO and its role in shaping meiotic recombination landscape.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hela Cell

SUBMITTER: Román González-Prieto

LAB HEAD: Roman Gonzalez Prieto

PROVIDER: PXD047332 | Pride | 2024-05-31

REPOSITORIES: Pride

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Project description: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 E2 ubiquitin-conjugating 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, a very promiscuous E2 enzyme that affects a broad range of processes. Here, we set out to identify in vivo targets of UBE2D3 by using SILAC-based quantitative ubiquitin diGly proteomics to study global proteome and ubiquitinome changes associated with UBE2D3 depletion. UBE2D3 depletion changed the global proteome, with 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 and, interestingly, identified pathways related to mRNA translation as top pathways being affected. Indeed, we find that ubiquitination of the ribosomal proteins RPS10 and RPS20, critical for ribosome-associated protein quality control (RQC), is dependent on UBE2D3. Moreover, we find that UBE2D3 limits monoubiquitination of histone H2B at lysine 120, mediated by the E3 ligase RNF20. This identifies UBE2D3 as a potential regulator of the diverse and critical cellular processes that are controlled by H2B monoubiquitination. Collectively, our findings show that depletion of an E2 ubiquitin-conjugating 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. Together with our identification of RQC factors and H2B as novel in vivo targets of UBE2D3, our work provides an important resource for further studies on the in vivo functions of UBE2D3.

Project description:We developed native elongating transcript sequencing (NET-seq, Churchman and Weissman Nature 2011, PMID: 21248844) combined with RNase footprinting of nascent transcripts (RNET-seq) to visualize translocation dynamics and nascent transcript errors in paused RNA polymerases in E. coli. We employed RNET-seq to the wild-type (WT) E. coli strain and to an isogenic strain deficient in genes for GreA and GreB (ΔgreAB). Gre factors and their eukaryotic analog TFIIS rescue backtracked complexes of RNAP. Briefly, the cells were rapidly lysed via spheroplasting, and the transcribing RNAPs were released from the genomic DNA by digestion with DNase I. Any ribosomes involved in co-transcriptional translation were separated from RNAP by digestion with RNase A. All RNAPs including those associated with the fragmented double-stranded DNAs and their 5’-truncated nascent RNAs were immobilized on Ni2+-NTA beads through the hexa-histidine-tagged β’ subunit and then extensively washed with a high-salt buffer. The 5’ ends of the transcripts in ECs were trimmed with RNase T1/V1 to leave a minimal length of RNA protected by RNAP. The RNases were subsequently removed by further washing of the beads. Elution with imidazole generated ECs carrying ~6-30 nt long transcripts. The nascent RNAs isolated from the ΔgreAB strain were longer than those from the WT strain and peaked at 18 nt versus 16 nt suggesting an enrichment of backtracked ECs, which is expected to occur in the absence of Gre-dependent 3’ RNA cleavage. The RNET-seq method involves trimming of excess nascent RNA from the 5’ ends leaving only the nascent RNA that is protected by RNAP. A previous in vitro study showed that an RNAP forming an EC protects different lengths of the 3’-proximal transcript from trimming by RNases A and T1 depending on the EC translocation state. Post-translocated, pre-translocated, and backtracked complexes protect 14-nt, 15-nt and >15-nt segments of the RNA, respectively. Because the very 3’ end of the RNA is extruded to a narrow pore within front of the enzyme during backtracking, the extruded RNA remains inaccessible to RNases increasing in length as backtracking increases. Thus, paused RNAP in either the pre- and post-translocated states as well as in different backtracked distances were monitored over the entire genome. The unique properties of our RNET-seq approach provided an opportunity to dissect the core mechanisms of different types of pausing in living cells.

			Action	DRS
	HUMAN.fasta	Fasta
	L20212000496a.raw	Raw
	L20212000497a.raw	Raw
	L20212000498a.raw	Raw
	L20212000499a.raw	Raw

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RNF212B E3 ligase is essential for crossover designation and maturation during male and female meiosis in the mouse

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