Interaction of hnRNP A1 with telomere DNA G-quadruplex structures studied at the single molecule level

Krüger, Asger Christian; Raarup, Merete K.; Nielsen, Morten Muhlig; Kristensen, Michael; Besenbacher, Flemming; Kjems, Jørgen; Birkedal, Victoria

doi:10.1007/s00249-010-0587-x

Cited by 32 publications

(32 citation statements)

References 33 publications

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“…Single-molecule FRET measurements were conducted using an inverted wide-field optical microscope and alternate laser excitation of the fluorescent donor (Cy3) and acceptor (Cy5); the setup has been described in more detail in Kruger et al (2010). Fluorescence was collected through the objective in epi geometry and sent to a CCD (MicroMax; Roper scientific) for detection.…”

Section: Methodsmentioning

confidence: 99%

Role of the primer activation signal in tRNA annealing onto the HIV-1 genome studied by single-molecule FRET microscopy

Beerens

Jepsen

Nechyporuk-Zloy

et al. 2013

RNA

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HIV-1 reverse transcription is primed by a cellular tRNAlys3 molecule that binds to the primer binding site (PBS) in the genomic RNA. An additional interaction between the tRNA molecule and the primer activation signal (PAS) is thought to regulate the initiation of reverse transcription. The mechanism of tRNA annealing onto the HIV-1 genome was examined using ensemble and single-molecule Förster Resonance Energy Transfer (FRET) assays, in which fluorescent donor and acceptor molecules were covalently attached to an RNA template mimicking the PBS region. The role of the viral nucleocapsid (NC) protein in tRNA annealing was studied. Both heat annealing and NC-mediated annealing of tRNAlys3 were found to change the FRET efficiency, and thus the conformation of the HIV-1 RNA template. The results are consistent with a model for tRNA annealing that involves an interaction between the tRNAlys3 molecule and the PAS sequence in the HIV-1 genome. The NC protein may stimulate the interaction of the tRNA molecule with the PAS, thereby regulating the initiation of reverse transcription.

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Section: Methodsmentioning

confidence: 99%

Role of the primer activation signal in tRNA annealing onto the HIV-1 genome studied by single-molecule FRET microscopy

Beerens

Jepsen

Nechyporuk-Zloy

et al. 2013

RNA

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“…and remains predominantly in a compact state when complexed with hnRNP A1 (Kruger et al, 2010). This finding is in contrast to the previously reported crystal structures of UP1-telomere DNA complexes (Ding et al, 1999), where the DNA oligonucleotide within the protein-DNA complex is in a fully open conformation.…”

Section: Telomere Extension By Telomerasecontrasting

confidence: 99%

“…1.7) (Wang et al, 2011). In addition, Quan Wang and his colleagues have shown that G-quadruplexes tend to form at the farthest 3´ end of telomere DNA (Wang et al, 2011, Kruger et al, 2010. The addition of telomeres using a minimum of four TTAGGG repeats can be extende by telomerase, while shorter than 4 repeats pose no extension (Zahler et al, 1991).…”

Section: G-quadruplex Structuresmentioning

confidence: 99%

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

Saig¹

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Telomere shortening leads to eukaryotic cell senescence, whereas enhanced telomerase activity is associated with telomere expansion and growth of cancer cells. Several studies have suggested hnRNPs are important for telomere maintenance including their ability to bind telomeres, and telomerase. The hnRNPs A/B family are highly abundant multifunctional proteins that bind to single-stranded DNA and RNA and perform many roles in cellular regulation. In this study, affinity pull-down assays, biosensor and UV-cross-linking studies were used to confirm that recombinant hnRNPs A2 and A3, compared to recombinant hnRNP A1, specifically interact with single-stranded telomeric DNA repeat TTAGGG and hexamer repeat. Tandem RRMs are required for strong binding, which is little changed by exclusion of the glycine-rich domain (hnRNPs Δ GRD).Additionally, TRAP assays, that indicate telomerase activity, showed that hnRNPs A1, A2and A3 bind to telomerase in cell extracts. It is not clear whether this association is mediated by binding a specific nucleotide region within telomerase RNA, or a protein component such as dyskerin, or both.In this study I have confirmed and refined the interaction site between the hnRNP A/B proteins and telomerase RNA (hTR). Serial UV-cross-linking RNA electrophoretic mobility shifting assays (REMSA) have shown that hnRNP A2 binds specifically with to a 17nt region within the RNA telomerase template (hTR). Deletion analysis of the hnRNP A/Bs revealed that these proteins bind preferentially to the hTR segment encompassing nucleotides hTR 57-72 mediated mainly through the RRM1 sequence. Importantly, I found that hnRNP A2 can interact simultaneously with telomeric ssDNA repeats. Both hnRNP A1 and hnRNP A3 showed no binding to the short segments of hTR in this assay, yet they could form a complex with full-length hTR. This difference can be explained by the increased insolubility of A1 and A3. Additionally, this study has indicated that both RRMs of hnRNP A2 are essential for either DNA-protein, or RNA-protein interactions, or both for simultaneous binding. In the chromatography experiments, RRM1 and RRM2 on their own were not able to mediate a simultaneous binding with both telomeric DNA and telomerase RNA, while short variant UP2 was successful to maintain this kind of binding feature.The results suggest that hnRNP A2 may help recruit telomerase to the ends of chromosomes.These hnRNP A/B family members are highly abundant in the cell nucleus, perform many roles in the cell and potentially influence telomere biogenesis. From these studies a model was proposed where the hnRNP A2 binding site is adjacent, or close to the template segment of hTR. The close proximity of the hnRNP A2 binding site to the template region on hTR and necessarily, to the end of the ssDNA 3' overhang, supports the proposal that these proteins play an active role in the addition of new telomeric repeats for cellular maintenance. VI Publications included in this thesisNo publications included. Contributions by others to the t...

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“…Therefore, if telomeric G-quadruplex forms in vivo, as has been proposed (6,(52)(53)(54), cellular mechanisms must exist to promote unfolding of this structure during telomere extension by telomerase. POT1 and proteins from the hnRNP family, like hnRNP A1, are able to disrupt G-quadruplex (55)(56)(57). However, these proteins also bind with high affinity to and occlude the 3′ end of the telomere, which could limit access of telomerase to telomere ends and inhibit telomere extension.…”

Section: Hnrnp A2* Localizes To the Nuclear Matrix And Associates Withmentioning

confidence: 99%

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

Wang

Tang

Zeng

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Interaction of hnRNP A1 with telomere DNA G-quadruplex structures studied at the single molecule level

Cited by 32 publications

References 33 publications

Role of the primer activation signal in tRNA annealing onto the HIV-1 genome studied by single-molecule FRET microscopy

Role of the primer activation signal in tRNA annealing onto the HIV-1 genome studied by single-molecule FRET microscopy

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

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

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