Abstract
Detection of low-affinity or transient interactions can be a bottleneck in our understanding of signaling networks. To address this problem, we developed an arrayed screening strategy based on protein complementation to systematically investigate protein-protein interactions in live human cells, and performed a large-scale screen for regulators of telomeres. Maintenance of vertebrate telomeres requires the concerted action of members of the Telomere Interactome, built upon the six core telomeric proteins TRF1, TRF2, RAP1, TIN2, TPP1, and POT1. Of the ∼12,000 human proteins examined, we identified over 300 proteins that associated with the six core telomeric proteins. The majority of the identified proteins have not been previously linked to telomere biology, including regulators of post-translational modifications such as protein kinases and ubiquitin E3 ligases. Results from this study shed light on the molecular niche that is fundamental to telomere regulation in humans, and provide a valuable tool to investigate signaling pathways in mammalian cells.
Highlights
From the ‡State Key laboratory for Biocontrol, Sun Yat-Sen University, Guangzhou, P
Work from our lab and others suggest that TPP1, along with POT1, TIN2, TRF1, TRF2, and RAP1, form a higher order complex at the telomeres (24 –27)
We have developed a high-throughput protein-protein interaction screening strategy based on the principle of the yellow fluorescent protein (YFP)1-based protein complementation assay (PCA/bimolecular fluorescent complementation (BiFC)) (52, 54 –56)
Summary
Genome-wide YFP Fluorescence Complementation Screen Identifies New Regulators for Telomere Signaling in Human Cells*□S. Detection of low-affinity or transient interactions can be a bottleneck in our understanding of signaling networks To address this problem, we developed an arrayed screening strategy based on protein complementation to systematically investigate protein-protein interactions in live human cells, and performed a large-scale screen for regulators of telomeres. Telomeres and telomerase help to ensure genome integrity in eukaryotes by enabling complete replication of the ends of linear DNA molecules, and preventing chromosomal rearrangements or fusion. For dividing cells such as stem cells and the majority of cancer cells, the telomerase is an essential positive regulator of their telomere length and determines the proliferative potential of these cells. Our findings provide a high-resolution map of the telomere interactome [19, 59, 60], and should greatly facilitate further studies of telomere signaling
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