Functional Diversity of Human Protection of Telomeres 1 Isoforms in Telomere Protection and Cellular Senescence

Abstract

Protection of telomeres 1 (POT1) proteins in various organisms bind telomeres and regulate their structure and function. In contrast to mice carrying two distinct POT1 genes encoding two POT1 proteins (POT1a and POT1b), humans have the single POT1 gene. In addition to full-length POT1 protein (variant v1), the human POT1 gene encodes four other variants due to alternative RNA splicing (variants v2, v3, v4, and v5), whose functions are poorly understood. The functional analyses of the NH 2 -terminally and COOH-terminally truncated POT1 variants in this study showed that neither the single-stranded telomere-binding ability of the NH 2 -terminal oligonucleotide-binding (OB) folds nor the telomerasedependent telomere elongation activity mediated by the COOH-terminal TPP1-interacting domain was telomere protective by itself. Importantly, a COOH-terminally truncated variant (v5), which consists of the NH 2 -terminal OB folds and the central region of unknown function, was found to protect telomeres and prevent cellular senescence as efficiently as v1. Our data revealed mechanistic and functional differences between v1 and v5: (a) v1, but not v5, functions through the maintenance of telomeric 3 ¶ overhangs; (b) p53 is indispensable to v5 knockdown-induced senescence; and (c) v5 functions at only a fraction of telomeres to prevent DNA damage signaling. Furthermore, v5 was preferentially expressed in mismatch repair (MMR)-deficient cells and tumor tissues, suggesting its role in chromosome stability associated with MMR deficiency. This study highlights a human-specific complexity in telomere protection and damage signaling conferred by functionally distinct isoforms from the single

Distinct Functions of POT1 at Telomeres

“…In agreement, TIFs are reduced in murine fibroblasts in which both POT1 genes are disrupted upon expression of human POT1 and TPP1 (21), overexpression of a truncated TPP1 protein defective in POT1 binding (TPP1 ⌬RD ) can induce TIFs (17), and lastly, knockdown of the v5 splice version of POT1 that does not interact with TPP1 induced fewer DNA damage foci than did knockdown of the predominant splice form that binds TPP1 (54). Mechanistically, we show that knockdown of POT1 results in an accumulation of RPA at telomeres, as evidenced by an increased number of STAR-positive cells.…”

POT1 Association with TRF2 Regulates Telomere Length

“…Third, the POT1-TPP1 heterodimer recruits telomerase to telomeres and promotes its processivity (72,73). As an entrée to studying how POT1 regulates telomere length in telomerase-positive cells, we note that expression of POT1 ⌬OB , a mutant lacking the N-terminal OB folds responsible for telomeric ssDNA binding (3,50,51), is one of the most potent means of inducing elongation of telomeres (2,50,51,74). This mutant clearly disrupts at least one critical aspect of telomere length regulation.…”

“…Telomeric ssDNA is bound directly by the protein POT1 (4, 5, 23, 52). POT1 acts in a heterodimer with the protein TPP1; this heterodimer promotes POT1 binding to telomeric ssDNA at least in vitro (72, 73), protects telomeres from being detected as sites of DNA damage (3,24,29,37,73,74), and inhibits replication protein A (RPA) localization to telomeres (3). In vitro-transcribed and -translated POT1 has also been shown to associate with recombinant glutathione S-transferase-TRF2 (75).…”

“…Taken together, these data suggest that POT1 also associates with TRF2, potentially directly, and this interaction facilitates POT1 association with telomeric chromatin (3,37,53,75). All the aforementioned proteins are found in larger complexes containing other proteins involved in telomere length regulation, DNA damage response, and sister chromatid association (13,16,22,49,56,57,60,67).Inhibition of any of the proteins that directly bind telomeric DNA (TRF1, TRF2, or POT1) or many of their interacting partners (TPP1, TIN2, or RAP1) in telomerase-positive human cells results in telomere elongation (10,38,43,47,51,56,66,69,71,74,77). Such inhibition has also been reported to disrupt telomere protein complexes (57) and induce the mislocalization of other telomere-associated proteins (37,39,50,51,75,77).…”

“…POT1 ⌬OB retains the protein interaction domain of the wildtype protein, which mediates its interaction with both TPP1 and TRF2 (3,39,50,74,75,77), and elongates telomeres in a dominant-negative fashion (2,50,51,74). This suggests that one of these protein interactions, when uncoupled from telomeric ssDNA binding, underlies the ability of POT1…”