MAD2L1BP Human

What is MAD2L1BP and what is its molecular function in human cells?

MAD2L1BP, also known as p31 comet, functions as a critical component of the spindle assembly checkpoint (SAC) machinery. This protein acts as an adaptor that binds mitosis arrest deficient 2 (MAD2) and recruits the AAA+-ATPase TRIP13 to disassemble the mitotic checkpoint complex (MCC) . This interaction is essential for cell-cycle progression after the spindle assembly checkpoint is satisfied. Structurally, MAD2L1BP harbors highly conserved central 'α-helix'- and flanked 'β-sheet'-organized domains that structurally mimic MAD2 and physically interact with it at the MAD2 dimerization interface . The C-terminal region, particularly the C-terminal 22 amino acids that fold into β9 and β10 strands, is crucial for its physical interaction with MAD2 .

How is MAD2L1BP expression regulated during oocyte maturation?

MAD2L1BP mRNAs are highly abundant in both human and mouse oocytes, particularly in those at advanced stages of maturation . This high expression level indicates an important role of MAD2L1BP in oocyte meiotic maturation in mammals. Similar to MAD2L1BP, the mRNA expression levels of MAD2 are also high in both human and mouse oocytes . This coordinated expression pattern suggests a tightly regulated expression mechanism to ensure proper stoichiometry of components involved in the spindle assembly checkpoint during oocyte maturation.

What is the relationship between MAD2L1BP and the mitotic checkpoint complex?

MAD2L1BP plays a crucial role in the disassembly of the mitotic checkpoint complex (MCC). The MCC is timely assembled and disassembled through the conformational transition of 'O-MAD2' to 'C-MAD2' via a 'MAD2 template' model throughout the cell cycle . The disassembly of the MCC machinery is predominantly achieved through MAD2L1BP, which acts as an adaptor that binds MAD2 and recruits ATPase TRIP13 to the MCC . This disassembly process is essential for cell cycle progression once the spindle assembly checkpoint is satisfied. Disruption of this MAD2- MAD2L1BP- TRIP13 axis leads to sustained SAC activation and consequently arrest of cell division .

What types of MAD2L1BP variants have been identified in human patients with fertility issues?

Research has identified several biallelic variants in MAD2L1BP associated with female infertility due to oocyte maturation arrest. These include:

• A homozygous p.R285* variant causing loss of C-terminal 22 amino acids

• Compound heterozygous variants including p.F173Sfs4* and c.21-94G>A (rs142226267)

• A homozygous p.R181* variant resulting in loss of 126 amino acids at the C-terminus

These variants were identified through whole-exome sequencing (WES) in three families with female patients diagnosed with primary infertility due to oocyte metaphase I (MI) arrest . The variants were validated as pathogenic through in silico prediction tools including SIFT, PolyPhen-2, MutationTaster, NNSplice, and ASSP .

What techniques can be used to evaluate the functional impact of MAD2L1BP variants?

Several experimental approaches can be used to assess the functional consequences of MAD2L1BP variants:

• mRNA expression analysis: Quantitative PCR (qPCR) can be used to measure the expression levels of MAD2L1BP mRNA in patient samples compared to controls .

• In vitro splicing assays: For variants potentially affecting splicing, researchers can synthesize plasmids comprising exons and flanking intronic sequences, transfect them into cells, and sequence the transcribed spliced mRNA products .

• Protein-protein interaction studies: Co-immunoprecipitation (Co-IP) assays using tagged MAD2 and wild-type or mutant MAD2L1BP constructs can assess the impact of mutations on protein binding capacity .

• Structural analysis: Crystal structure analysis (e.g., using PDB data like 2QYF) can help determine how mutations affect the structural domains important for protein function .

• Mouse oocyte functional assays: Microinjection of wild-type or mutant MAD2L1BP cRNAs into mouse oocytes followed by monitoring oocyte maturation can provide functional evidence of variant pathogenicity .

How can researchers design rescue experiments to validate MAD2L1BP variant pathogenicity?

Rescue experiments provide powerful evidence for variant pathogenicity in oocyte maturation defects. A methodological approach includes:

• Patient oocyte collection: Obtain oocytes from patients with MAD2L1BP mutations following informed consent .

• cRNA preparation: Synthesize wild-type MAD2L1BP cRNA in vitro for microinjection .

• Microinjection procedure: Inject approximately 5 pl of MAD2L1BP cRNA solution (1000 ng/μl) into patient oocytes .

• In vitro maturation: Culture injected oocytes under appropriate conditions for in vitro maturation .

• Phenotype assessment: Monitor polar body extrusion (PBE) as an indicator of successful meiosis I completion .

In the research presented, this approach successfully rescued oocyte maturation in patient samples, with 4 out of 6 oocytes from a patient with p.R285* MAD2L1BP variant extruding the first polar body after supplementation with wild-type MAD2L1BP cRNA, while control oocytes with sham injection failed to mature .

What methodological approaches can be used to investigate MAD2L1BP function in mouse models?

Several experimental approaches using mouse models can help investigate MAD2L1BP function:

How does the molecular mechanism of MAD2L1BP function differ between mitosis and meiosis?

The function of MAD2L1BP in disassembling the mitotic checkpoint complex appears to be conserved between mitosis and meiosis, but with some important distinctions:

• In mitotic cells, MAD2L1BP deficiency leads to mitotic delay and lagging chromosomes, but some cells manage to reinitiate the cell cycle .

• In contrast, oocytes with MAD2L1BP mutations show complete meiotic arrest at metaphase I .

This difference suggests that meiotic cells may have stricter requirements for proper SAC regulation than mitotic cells. The extended duration of meiosis compared to mitosis, particularly the prolonged metaphase I arrest that can last for years or decades in humans, might explain the heightened sensitivity of oocytes to MAD2L1BP dysfunction. Further research comparing the molecular dynamics of MAD2L1BP interactions in mitotic versus meiotic cells could help elucidate these differences.

What are the potential therapeutic approaches for treating infertility caused by MAD2L1BP mutations?

The successful rescue of oocyte maturation through microinjection of wild-type MAD2L1BP cRNA into patient oocytes suggests a potential therapeutic avenue . This approach demonstrates that supplementation with functional MAD2L1BP can overcome the meiotic arrest caused by mutant protein.

Potential therapeutic strategies might include:

• cRNA or mRNA supplementation: Direct microinjection of wild-type MAD2L1BP mRNA into oocytes during in vitro maturation procedures .

• CRISPR-based approaches: Gene editing to correct pathogenic variants in affected individuals' germline or in embryos.

• Protein supplementation: Development of methods to deliver functional MAD2L1BP protein directly to oocytes.

• Small molecule modulators: Identification of compounds that could bypass the need for functional MAD2L1BP by directly influencing downstream pathway components.

The rescue experiments performed in the study provide proof-of-concept that restoring MAD2L1BP function can reverse the meiotic arrest phenotype, opening possibilities for therapeutic interventions in affected individuals .

How does the transcriptome change in oocytes with MAD2L1BP mutations?

While the complete transcriptomic analysis of MAD2L1BP-mutant oocytes wasn't fully detailed in the search results, the research suggests that MAD2L1BP mutations disrupt the MAD2- MAD2L1BP- TRIP13 axis, leading to sustained SAC activation . This disruption likely influences the expression of numerous genes involved in cell cycle regulation, chromosome segregation, and oocyte maturation.

A comprehensive transcriptomic analysis of mutant versus wild-type oocytes would be valuable to:

• Identify dysregulated pathways beyond the immediate SAC components

• Discover potential compensatory mechanisms that might be activated

• Reveal novel biomarkers for oocyte maturation defects

• Identify potential therapeutic targets that could be modulated to overcome meiotic arrest

Such analysis would require single-cell RNA sequencing approaches given the limited number of oocytes available from patients with MAD2L1BP mutations.

How does MAD2L1BP research connect to other genes involved in human infertility?

MAD2L1BP research provides important connections to other genes involved in human infertility, particularly those in the spindle assembly checkpoint pathway:

• TRIP13: Recent studies have identified pathogenic biallelic variants in TRIP13 causing female primary infertility due to oocyte maturation arrest predominantly at the MI stage . Since MAD2L1BP recruits TRIP13 to disassemble the MCC, mutations in either gene can lead to similar phenotypes.

• CDC20: Deleterious variants in CDC20, a core component of MCC, have been discovered accounting for human oocyte meiotic arrest and early embryonic failure .

These findings collectively establish the importance of the spindle assembly checkpoint pathway in human fertility and suggest that other components of this pathway could be candidates for genetic screening in infertility cases with oocyte maturation arrest.

What are the current limitations in studying MAD2L1BP function in human reproduction?

Several key limitations exist in the study of MAD2L1BP in human reproduction:

• Limited patient samples: The research identified MAD2L1BP variants in only a small cohort of 50 oocyte MI-arrested patients . Larger cohorts would provide more statistical power.

• Oocyte availability: The limited number of oocytes available from affected individuals restricts the scope of functional studies that can be performed .

• Animal model challenges: MAD2L1BP-deficient mice die perinatally due to neonatal hypoglycemia, making it difficult to study reproductive phenotypes in homozygous knockout animals .

• Technical challenges: Standard knockdown approaches like siRNA have shown limitations in oocytes, as demonstrated by the failure to significantly deplete MAD2L1BP mRNA in mouse oocytes despite effectiveness in somatic cells .

• Complexity of meiotic regulation: The intricate regulatory network governing meiotic progression makes it challenging to isolate the specific effects of MAD2L1BP dysfunction from secondary consequences.

Addressing these limitations will require innovative approaches, including the development of conditional or tissue-specific knockout models, improved methods for manipulating gene expression in oocytes, and more efficient techniques for phenotypic analysis of limited patient samples.

What is the impact of different MAD2L1BP variants on protein function?

This table summarizes the functional consequences of different MAD2L1BP variants identified in patients with oocyte maturation arrest .