Dataset Information

PRP4 is a spindle assembly checkpoint protein required for MPS1, MAD1, and MAD2 localization to the kinetochores.

ABSTRACT: The spindle checkpoint delays anaphase onset until every chromosome kinetochore has been efficiently captured by the mitotic spindle microtubules. In this study, we report that the human pre-messenger RNA processing 4 (PRP4) protein kinase associates with kinetochores during mitosis. PRP4 depletion by RNA interference induces mitotic acceleration. Moreover, we frequently observe lagging chromatids during anaphase leading to aneuploidy. PRP4-depleted cells do not arrest in mitosis after nocodazole treatment, indicating a spindle assembly checkpoint (SAC) failure. Thus, we find that PRP4 is necessary for recruitment or maintenance of the checkpoint proteins MPS1, MAD1, and MAD2 at the kinetochores. Our data clearly identify PRP4 as a previously unrecognized kinetochore component that is necessary to establish a functional SAC.

SUBMITTER: Montembault E

PROVIDER: S-EPMC2080909 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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PRP4 is a spindle assembly checkpoint protein required for MPS1, MAD1, and MAD2 localization to the kinetochores.

Montembault Emilie E Dutertre Stéphanie S Prigent Claude C Giet Régis R

The Journal of cell biology 20071112 4

The spindle checkpoint delays anaphase onset until every chromosome kinetochore has been efficiently captured by the mitotic spindle microtubules. In this study, we report that the human pre-messenger RNA processing 4 (PRP4) protein kinase associates with kinetochores during mitosis. PRP4 depletion by RNA interference induces mitotic acceleration. Moreover, we frequently observe lagging chromatids during anaphase leading to aneuploidy. PRP4-depleted cells do not arrest in mitosis after nocodazol ...[more]

PMID: 17998396

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Project description:Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle checkpoint which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1D or mad2D strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrest Cryptococcus cells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effective in vitro substrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. We conclude that CnMps1 protein kinase, Mad1 and Mad2 proteins have all conserved their important, spindle checkpoint signalling roles helping ensure high fidelity chromosome segregation.

Dataset Information

PRP4 is a spindle assembly checkpoint protein required for MPS1, MAD1, and MAD2 localization to the kinetochores.

Publications

PRP4 is a spindle assembly checkpoint protein required for MPS1, MAD1, and MAD2 localization to the kinetochores.

Similar Datasets

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