Title: Structure and function of the telomerase inhibitor PinX1
Description: Maintenance of telomere homeostasis by the telomere-associated complex shelterin is critical in dictating the balance between cellular senescence and oncogenic growth. However, it is unclear how shelterin communicates with telomerase to regulate telomere lengths. PinX1 is a telomerase inhibitor and a putative tumor suppressor that was discovered through its interaction with one of the main shelterin components, TRF1. Here, we describe the characterization of the TRF1-PinX1 interaction and demonstrate that TRF1 recruitment of PinX1 to telomeres is necessary for PinX1-induced telomere shortening. Through mutational and structural analysis of the PinX1-TRF1 interface, we have determined a key residue necessary for anchoring PinX1 to TRF1, which is further required for PinX1 localization to telomeres and inhibition of telomere elongation. These findings support a role for PinX1 as a link between shelterin and telomerase towards inhibiting telomere elongation. To determine whether this role is deregulated in human cancers, and to investigate the role of PinX1 in oncogenesis, we sequenced the PINX1 gene from human breast cancer samples and identified several polymorphisms that cluster within the telomerase inhibitory and TRF1-interacting domain of PinX1. Currently, these PinX1 genetic alterations have not revealed any conclusive disruption in PinX1 telomerase inhibition activity, telomere localization or binding with TRF1 and telomerase. It is possible that these genetic variations regulate an as yet undetermined function of PinX1. In an effort to uncover other PinX1 functions, we performed a large-scale tandem affinity purification of PinX1 to identify novel interacting partners. Proteins identified in the screen suggested roles for PinX1 in RNA processing and DNA damage response.

We specifically validated the interaction between PinX1 and a DEAH-box RNA helicase, DHX15, and an E3 ubiquitin ligase, EDD1. These findings indicate a previously undefined function for PinX1 in the nucleolus, and reveal a possible mechanism for the tightly controlled regulation of PinX1 protein levels. Taken together, these studies demonstrate the complexity of PinX1 interactions, and highlight roles at telomeres and in the nucleolus that may elucidate its function as a tumor suppressor.