Proximity Labeling Identifies Cell Cycle Specific Interactions of Retinoblastoma Protein

Rashkovskii, Mikhail

Student Work

Proximity Labeling Identifies Cell Cycle Specific Interactions of Retinoblastoma Protein

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The Retinoblastoma tumor suppressor protein is crucial for a number of cellular functions majority of which relate to cell proliferation and genomic stability. While regulation of E2F- dependent gene expression by Rb had been mechanistically tied to cell proliferation and chromatin remodeling, mechanisms underlying other cellular functions of Rb remain unclear. To better understand the molecular interactions that drive Rb’s diverse functions, proximity-based labeling was performed using an Rb-TurboID fusion system, followed by mass spectrometry analysis of labelled proteins in asynchronous, G2, and M phase cells. Analysis of labeled proteins, combined with literature mining of physical interactions, sequence-based motif analysis, and STRING functional grouping allowed for the identification of 200 high interest proteins distributed among cell cycle conditions and 8 functional groups: splicing, ribosome, mitochondria, DNA, cytoskeleton, vesicles, ER/Golgi, apoptosis. Detailed investigation of DNA groups gave insights in molecular mechanisms behind Rb function in DNA damage response, chromatin remodeling, and chromosome segregation, and allowed the identification of new potential Rb interactors specific to cell cycle stages. The analysis of other functional groups suggest previously uncharacterized functions of Rb in mitochondrial TCA cycle in G2, ribosome biogenesis in G1/S and functioning in G2 and M, nuclear splicing throughout cell cycle and mRNA export in G2, as well as Rb glycosylation in Golgi mainly in G2 phase.

This report represents the work of one or more WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement. WPI routinely publishes these reports on its website without editorial or peer review.

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