Many proteins are involved in the synthesis and maintenance of telomeres. They include Pot1, which binds the G-rich telomere end (51
), and TRF1 and TRF2, which bind double-stranded telomeric DNA (16
). These proteins are negative regulators of telomere extension. Loss of TRF2 leads to cell-cycle arrest, apoptosis (52
) and end-to-end ligation of telomeres (53
). Other proteins associated with TRF1, TRF2 or the ssDNA repeats have been identified, including several members of the hnRNP family. The roles of the hnRNPs in telomere biology are not well established but if, as proposed, some recruit telomerase to the chromosome ends they would be expected to support telomere extension, with diminished protein expression resulting in telomere shortening.
The hnRNP A/B proteins are abundant in many cell types. They are localized primarily to the nucleus where they may bind the telomeric DNA repeat (24
) and participate in telomere maintenance. They are also involved in packaging nascent mRNA, in alternative splicing (24
), and in cytoplasmic RNA trafficking (44
), translation (57
) and stabilization (58
). In each role they presumably interact with sets of molecules that incorporate different RNA or DNA elements.
All the major proteins in the hnRNP A/B family, A1, A2 and A3, and their isoforms, were isolated in our pull-down experiments with the telomeric repeat. In the rat brain, hnRNPs A2 and A3 are the predominant binding proteins. These proteins share high sequence identity, with hnRNPs A3 and A1 being more closely related than hnRNPs A2 and A1. Over the tandem RRM region they have ~80% sequence identity and hence hnRNPs A2 and A3 are likely to share the typical RRM βαββαβ fold of hnRNP A1 (60
). The crystal structure of the UP1 proteolytic fragment of hnRNP A1 (residues 1–196 of the human protein) complexed to the single-stranded telomeric repeat [d(TTAGGG)2
) shows that the amino acid residues of hnRNP A1 that interact with the oligodeoxyribonucleotide are present in hnRNP A2, with the exception of a single Lys to Arg substitution. Because only the main chain atoms of this residue, K183, interact with G11, it is unlikely that substitution with arginine would interfere with the interaction between hnRNP A2 and the telomeric repeat. Despite this conservation of interacting residues, there are differences in the binding of the telomeric repeats by hnRNPs A1 and A2, as discussed below.
In an earlier study we presented evidence from EMSA and biosensor data that recombinant hnRNP A2 has two sites that bind oligoribonucleotides or oligodeoxyribonucleotides, but with this protein only the sequence-specific site was occupied in the presence of heparin (45
). In contrast, we discovered in this study that rat hnRNP A2 also has two sites but binds oligodeoxyribonucleotides at the non-specific site even in the presence of heparin (). Possession of two sites raises the possibility of the protein acting as a non-covalent cross-linker of nucleic acids. DNA or RNA molecules (containing e.g. the telomere repeat or A2RE11) occupying the specific site may be linked to any other ssDNA. The ability to bind the telomeric repeat at both sites may also be important for protection of the ssDNA from nuclease activity.
Although the telomere repeat binds the isolated RRM1 of hnRNP A2 weakly (if at all) and isolated RRM2 somewhat more strongly, the tandem extended RRMs (residues 1–189) are needed for binding that matches the whole protein. This binding pattern deviates from that for hnRNP A1, the first RRM of which was found to bind more tightly than RRM2 (28
). RRM1 is sufficient for A1/UP1binding to a telomeric DNA oligonucleotide (30
). This difference points to the divergence in the mode of interaction for hnRNPs A1 and A2, with the latter possibly not mimicking the anti-parallel dimeric molecular arrangement observed in the crystalline state, in which oligonucleotides bind RRM1 of one hnRNP A1 molecule and RRM2 of the other.
The requirements in the nucleic acid for binding to the sequence-specific site of hnRNP A2 were established by mutational analysis, starting with A2RE11. This led to a consensus sequence that includes the telomeric sequence () and the hnRNP A1-binding d(GGCAG) tandem repeats of the mouse hypervariable minisatelite (29
). Changes in a single nucleotide have been shown to markedly alter oligonucleotide binding to hnRNP A2 () and UP1 (27
). As a consequence of the redundancy at several positions in the consensus sequence, it can give rise to many different oligonucleotides. hnRNP A2, a recognized multi-tasking protein (61
), may therefore potentially interact with numerous different RNA or DNA elements, thereby influencing many metabolic or signaling pathways. Moreover, it could act as the link between pathways utilizing nucleic acids that compete for binding to its RRMs.
UP1 binds the telomeric repeat sequence specifically in vitro
), protecting it from degradation by nucleases, and demonstrates helix-unwinding ability (62
). UP1 also binds the telomerase holoenzyme in vitro
, promoting telomere extension at low concentrations (31
), but inhibiting it at higher concentrations (28
). In addition, both hnRNP A1 and UP1 bind telomerase RNA and telomeric DNA simultaneously in vitro
), and may thus be capable of recruiting telomerase to form part of the protective complex on telomere ends.
The segment of hnRNP A2 equivalent to UP1, UP1-B (which is contained within RRM1+2′), is necessary and sufficient to bind the telomeric sequence in vitro
but it did not mirror the behavior of UP1: in our experiments it did not protect the telomeric DNA (). Protection against endonuclease-catalyzed degradation required the whole protein, suggesting that the glycine-rich C-terminal domain of hnRNP A2 plays a critical role in this action (). Protection in vivo
is also provided by other telomere-associated molecules that bind to multiple sites, such as Pot1 (51
hnRNP A2 retarded the 5′ 71-nt segment of hTR in an EMSA (). This includes the template region which is within the single-stranded region (63
) that is most likely to bind hnRNP A2. The apparent binding of hnRNP A2 to dimers, rather than monomers, of the 5′ 71-nt hTR segment () may not faithfully recapitulate the molecular interactions in vivo
, but functional telomerase is dimeric and includes two RNA molecules that cooperate functionally (64
Fiset and Chabot (30
) earlier proposed that hnRNP A1 could simultaneously bind telomerase, through interaction with its RNA, and the telomeric DNA repeat, thus providing a mechanism for recruitment of telomerase to the chromosome ends (32
). Our observation that hnRNP A2 has two binding sites for single-stranded oligonucleotides leads to a parallel conclusion but, based on current evidence, the molecular mechanisms differ for these two proteins. The sites on hnRNP A1 for the telomeric DNA repeat and telomerase RNA were identified as residing on RRM1 and RRM2, respectively, whereas both RRM modules of hnRNP A2 are needed for tight binding to the repeat. RRM1 and the tandem RRMs of hnRNP A2 bind the 71-nt RNA segment, whereas no binding of RRM2 was apparent (). The telomerase RNA is therefore unlikely to be bound to the non-specific hnRNP A2 site, which appears not to bind oligoribonucleotides strongly (), nor does it bind the entire sequence-specific site, which spans the tandem RRMs. Further investigation is needed to establish whether its association with RRM1 makes use of part of the sequence-specific site.
In the mouse erythroleukemic cell line CB3 (66
), there is no measurable hnRNP A1 expression and the terminal repeat DNA fragments are markedly shorter than those in the similar cell line, CB7, which expresses hnRNP A1 (31
). But even after multiple passages of CB3, the mean terminal repeat fragment length did not change significantly, indicating that although hnRNP A1 may be required for full telomerase-mediated extension, in its absence another protein (or proteins) can prevent degradation of the telomere. On the basis of the results reported here, it is possible that the closely related hnRNP A2 protein can fulfill some or all of these functions.
Interestingly, hnRNP A2 and TRF2 are both present at higher than background levels in APBs. These intranuclear domains, which contain extrachromosomal telomeric DNA and telomere-specific binding proteins together with PML proteins, are specific for ALT-positive cell lines (11
). Their function is unknown, but it has been proposed that they might be depots of telomeric DNA and associated proteins required for ALT, or function as platforms for the ALT process. Telomeric DNA is not associated with the PML bodies of telomerase-positive cell lines and it is absent from many PML bodies within ALT cell nuclei, i.e. APBs are a subset of the PML bodies in ALT cells. The presence of hnRNP A2 in APBs (but not in PML bodies that do not contain telomeric DNA) suggests that telomeric chromatin is sufficient for the localization of hnRNP A2, and that hTR and telomerase activity are not required. hnRNP A2 was not present in every APB, consistent with the possibility that some aspect of the state of the telomeric chromatin is responsible for hnRNP A2 localization. It will be of interest to determine whether there are interactions among the hnRNPs and other proteins, such as Pot1 (51
), that protect the telomere ends from degradation.
In conclusion, from our studies it is evident that hnRNP A2 binds in a complex fashion to ssDNA and ssRNA. We found that rat brain hnRNP A2 has two sites for binding oligonucleotides. One binds oligodeoxyribonucleotides in preference to oligoribonucleotides and shows little discrimination between diverse sequences. The second, which requires both RRMs, has been shown to bind to sequences defined by the consensus sequence N(A,C,T)(C,T)(A,T)(A,G)G(C,G,T)(A,T)NNN: this sequence encompasses the telomeric repeat sequence. In addition, the template-containing segment of telomerase RNA binds to RRM1, raising the possibility that it completes with the ssDNA telomeric repeat for the hnRNP A2 sequence-specific site. As the G-rich overhanging strand lengthens, it may compete more effectively with telomerase RNA for binding to RRM1, displacing the RNA and thus limiting the telomerase-mediated strand extension. Although hnRNP A2 binds telomerase RNA, the preferential localization of hnRNP A2 with TRF2 in APBs suggests that telomerase is not needed for association with telomeric DNA. Combined with our in vitro binding data, the localization in APBs suggests an important role for this protein in telomere maintenance.