Telomere- and telomerase-interacting protein that unfolds telomere G-quadruplex and promotes telomere extension in mammalian cells

Abstract

Telomere extension by telomerase is essential for chromosome stability and cell vitality. Here, we report the identification of a splice variant of mammalian heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2), hnRNP A2*, which binds telomeric DNA and telomerase in vitro. hnRNP A2* colocalizes with telomerase in Cajal bodies and at telomeres. In vitro assays show that hnRNP A2* actively unfolds telomeric G-quadruplex DNA, exposes 5 nt of the 3′ telomere tail and substantially enhances the catalytic activity and processivity of telomerase. The expression level of hnRNP A2* in tissues positively correlates with telomerase activity, and overexpression of hnRNP A2* leads to telomere elongation in vivo. Thus, hnRNP A2* plays a positive role in unfolding telomere Gquadruplexes and in enhancing telomere extension by telomerase.H uman chromosome ends are protected by telomeres, which are composed of TTAGGG DNA repeats and associated proteins. Telomeres shorten with each cell division because of incomplete DNA-end replication (1). Cells compensate for telomere attrition through the action of telomerase, a specialized reverse transcriptase that adds telomeric repeats to the 3′ end of the telomere (2). Normal somatic cells do not express telomerase and undergo replicative senescence. The expression of telomerase is a hallmark of highly proliferative stem cells and most cancer cells. In budding yeast, the recruitment of telomerase to the telomere end is mediated by Cdc13 and Est1. Cdc13 is a singlestranded telomere DNA-binding protein that associates with Est1, a protein that interacts with the RNA component of yeast telomerase (3,4). Similarly, in Tetrahymena, Teb1 bridges the interaction between telomerase and the telomere, which promotes highly processive telomere extension by telomerase (5). However, the mechanism(s) and factors required to promote a similar interaction between the telomere and telomerase in mammalian cells are poorly understood.Telomere DNA can adopt a four-stranded G-quadruplex structure (6) that can be either intermolecular and intramolecular. Although an intermolecular G-quadruplex is an excellent substrate for ciliate telomerases (7), an intramolecular G-quadruplex is not. In vertebrates, intramolecular G-quadruplexes (hereinafter referred to as G-quadruplex) preferentially form at the furthest 3′ end of the telomeric DNA (8), rendering it inaccessible to telomerase. As a result, this structure inhibits telomere extension (9-11). Only a few proteins have been identified that can disrupt Gquadruplex. One such protein is protection of telomeres 1 (POT1) (12), a component of the telomere shelterin complex that binds telomere overhangs with high affinity (13). Despite its ability to disrupt G-quadruplex, POT1 actually inhibits telomere extension by binding to the telomere overhang and blocking telomerase access to the overhang (14-16).Some proteins of the hnRNP family are also able to unfold telomeric G-quadruplex (17, 18). These proteins interact with telomeric ssDNA (19) and telomerase in vitro (20...

Profile of POT1 as telomerase shelterin component discriminatesbetween cervical cancer and normal cervical cells

“…It may indicate that T-loop stabilization provided by POT1 serves to prevent excessive telomere elongation by telomerase. G-quadruplex disruption will inhibit telomerase access to the telomere end (9). POT1 may block the action of telomerase (8); on the other hand, telomerase recruitment and elongation of telomeres were preceded by POT1 action (10,11).…”

Role of hnRNP A/B proteins in telomere maintenance and telomerase activity

“…Thus, hnRNP A2 * plays a positive role in unfolding telomere G-quadruplexes and in enhancing telomere extension with telomerase (Fig 1.8) (Wang et al, 2012). hnRNP A2/B1…”

“…This could also be true for other abundant hnRNPs (Dallaire et al, 2000, Huang et al, 2008, Tanaka et al, 2007. hnRNP A2 * may be unique in its ability to actively unfold telomere Gquadruplexes while leaving 3' telomere ends accessible (Wang et al, 2012). A single-stranded telomere overhang forms intramolecular G-quadruplex.…”

“…The first evidence for the association of hnRNPs with telomeric DNA was obtained during attempts to isolate mammalian homologs of the Oxytricha telomere end-34 binding protein (On-TEBP) from mouse liver nuclear extracts (Ishikawa et al, 1993, Mckay andCooke, 1992). Since then, hnRNPs A1 (Labranche et al, 1998, Choi et al, 2012, hnRNP A2/B1 (Kamma et al, 2001, Moran-Jones et al, 2005, Wang et al, 2012, hnRNP A3 , hnRNP D (Enokizono et al, 2005, Eversole andMaizels, 2000), hnRNP E (Ford et al, 2002) and hnRNP K (Denisenko and Bomsztyk, 2002), Moreover, mouse cell lines deficient in hnRNP A1 expression are characterized by shorter telomeres than a related mouse cell line expressing normal levels of hnRNP A1 protein (Labranche et al, 1998). Ford and colleagues (2002) have proposed a model whereby hnRNPs bind telomeric single-stranded overhangs and recruit telomerase activity for telomere extension (Fig 1.12).…”

“…Telomerase does not extend the telomeric G-quadruplex unless it carries a single-stranded 3´ tail of at least 8 nt (Wang et al, 2011). During excessive extension, hnRNP A2 may intercept newly synthesized telomeric repeats, preventing them from forming G-quadruplexes, which consequently deters telomerase (Wang et al, 2012).…”