Mechanistic Basis for CENP-32 Mediated Regulation of Cell Division

In the human body, trillions of cells undergo division every day. During each division, the genetic information which is in the form of chromosomes need to be equally and identically distributed to the newly formed daughter cells. Distribution of chromosomes is achieved by an elaborate machinery called the mitotic spindle. In humans, the mitotic spindle is formed by a filamentous network called microtubules which are organised by an organelle called centrosome. During cell division cells possess two centrosomes that move away from each other and orchestrate microtubule network to establish the mitotic spindle. The centrosomes form the opposite ends of the mitotic spindle where microtubule networks converge and are called spindle poles. Physical attachment of centrosomes to the spindle poles is crucial for distributing chromosomes and centrosomes accurately to the daughter cells. Defective distribution of chromosomes and/or centrosomes are associated with several human health disorders such as cancer, microcephaly and primordial dwarfism. Hence understanding how centrosomes are attached to the spindle poles is important to better understand the related medical conditions and to find a possible cure.

The proposed work focuses on an essential human protein called CENP-32, which when removed from cells results in the detachment of centrosomes from spindle poles affecting the integrity of the mitotic spindle. CENP-32 mutations have also been found in patients with neurodevelopmental disorders such as microcephaly, seizures and developmental delays. The mitotic spindle lacking centrosomes at the poles is not capable of distributing the chromosomes accurately and as a consequence will result in daughter cells with inappropriate chromosome and/or centrosome numbers, a condition often associated with cancer and developmental disorders. Proteins exert their function by interacting with and/or modifying biomolecules including proteins and nucleic acids (RNA and DNA). Proteins acquire their function through their three-dimensional structure which provides them their ability to interact with/modify other biomolecules. To understand how CENP-32, a likely RNA binding protein, ensures the physical attachment of centrosomes to spindle poles, we propose: (1) to study the structure of CENP-32 and identify the RNA modifications it makes, (2) to identify the proteins/RNA that CENP-32 interacts with, (3) to delineate how RNA modifying activity of CENP-32 facilitate centrosome-spindle pole attachment, and (4) assess how CENP-32 patient mutations affect CENP-32 activity and function.

The outcome of this research will advance our understanding of how centrosomes help build an intact mitotic spindle essential for generating daughter cells with correct genetic information. CENP-32 is a protein essential for cell survival, hence the outcome of the proposed research will also pave way for exploring the possibility of blocking CENP-32 function in cancer cells and rectifying CENP-32 defect in patients with associated neurodevelopmental disorders.

The mitotic spindle is a microtubule-based apparatus responsible for segregating chromosomes and centrosomes to daughter cells during cell division. The bipolar structure of the mitotic spindle is formed by microtubules emanating from centrosomes that reside at opposite ends of the spindle forming the spindle poles. For error-free chromosome and centrosome segregation, centrosomes need to remain attached to spindle poles during mitosis. Centrosome detachment from spindle poles perturbs mitotic spindle integrity resulting in chromosome and centrosome segregation errors. Centrosome dysfunction and associated segregation errors are implicated in cancer, microcephaly and primordial dwarfism. Hence, understanding the mechanisms of centrosome-spindle pole attachment is of prime importance.

CENP-32, is a putative RNA methyltransferase essential for cell viability and centrosome-spindle pole attachment. Recent work from our collaborator has identified CENP-32 missense mutations in patients with neurodevelopment disorders. Here, we aim to obtain mechanistic understanding of how CENP-32 regulates centrosome-spindle pole attachment and how patient mutations affect CENP-32 function. Using an integrative structure-function analysis, we will: 1) characterise the structure and enzymatic activity of CENP-32, 2) identify cellular substrates and determine the structural basis for its substrate specificity, 3) elucidate the molecular basis for how CENP-32 activity regulates centrosome-spindle pole attachment using structure-guided mutants in cells, and 4) investigate how CENP-32 patient mutations affect the enzymatic activity and function of CENP-32.

The outcome will provide crucial insights into novel molecular pathways involving RNA modification/binding in regulating centrosome function and the mitotic spindle. Considering the direct disease relevance, this study will also open up new avenues for targeting CENP-32 to cure centrosome dysfunction related health disorders.