hnRNP A2, a potential ssDNA/RNA molecular adapter at the telomere

Abstract

The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multi-tasking protein that acts in the cytoplasm and nucleus. We have explored the possibility that this protein is associated with telomeres and participates in their maintenance. Rat brain hnRNP A2 was shown to have two nucleic acid binding sites. In the presence of heparin one site binds single-stranded oligodeoxyribonucleotides irrespective of sequence but not the corresponding oligoribonucleotides. Both the hnRNP A2-binding cis-acting element for the cytoplasmic RNA trafficking element, A2RE, and the ssDNA telomere repeat match a consensus sequence for binding to a second sequence-specific site identified by mutational analysis. hnRNP A2 protected the telomeric repeat sequence, but not the complementary sequence, against DNase digestion: the glycine-rich domain was found to be necessary, but not sufficient, for protection. The N-terminal RRM (RNA recognition motif) and tandem RRMs of hnRNP A2 also bind the single-stranded, template-containing segment of telomerase RNA. hnRNP A2 colocalizes with telomeric chromatin in the subset of PML bodies that are a hallmark of ALT cells, reinforcing the evidence for hnRNPs having a role in telomere maintenance. Our results support a model in which hnRNP A2 acts as a molecular adapter between single-stranded telomeric repeats, or telomerase RNA, and another segment of ssDNA.

Figure 1

Rat hnRNP A2 has sequence-specific and non-specific sites for binding oligodeoxyribonucleotides. A western blot of protein binding to oligodeoxyribonucleotides and oligoribonucleotides immobilized on superparamagnetic beads in pull-down experiments with brain protein extracts. Heparin was added to the protein extract to suppress non-specific binding. The protein was detected with a polyclonal primary antibody to a peptide from human/mouse hnRNP A2, and an alkaline-phosphatase-conjugated secondary antibody. These oligonucleotides were: the A2RE11 trafficking element (A2RE11 and dA2RE11, the former being the oligoribonucleotide), the AU-rich element (AURE and dAURE), the β-actin mRNA zipcode (ZIP and dZIP) and the non-specific sequence (44,46) (NS1 and dNS1). With the exception of the A2RE11, which binds to a specific site on hnRNP A2, the oligodeoxyribonucleotides but not the oligoribonucleotides bound hnRNP A2.

Figure 2

hnRNPs A1, A2, A3 and their isoforms are isolated in pull downs with a telomeric oligonucleotide. (A) Rat brain protein extract was incubated with superparamagnetic particles bearing no oligonucleotide (track 2), the scrambled A2RE ribonucleotide NS1 (track 3) or d(TTAGGG)₄ (track 4). Bound proteins were eluted using the SDS–gel electrophoresis sample preparation solution. Proteins were separated on a 15 cm 12% SDS–polyacrylamide gel and stained with Coomassie Blue R250. The four arrows on the right indicate the hnRNP A3 isoforms (47) and the arrowhead indicates hnRNP A2. The masses of standard proteins (track 1) are shown in kDa (the standards used in this experiment suggest that the slowest migrating hnRNP A3 band has a molecular mass over 45 kDa; however, it normally migrates with an apparent mass of 42 kDa). (B) Proteins isolated as above were separated by SDS–PAGE and electroblotted onto a polyvinylidine difluoride membrane. Antibodies directed against peptides in hnRNPs A1, A2/B1(labeled A2), B1, and the two higher molecular weight isoforms (A3^N) and all four isoforms of hnRNP A3 (A3^C) were used to demonstrate the presence of these proteins in the eluate (47). Molecular masses, in kDa, are indicated on the right.

Figure 3

The telomeric repeat competes for the hnRNP A2 dA2RE11 binding site. Competition UV-cross-linking EMSAs in which 1.5 pmol ³²P-labeled dA2RE11, dNS1 or telomeric sequences (Telo1 and Telo3) were incubated with 10 pmol of purified recombinant hnRNP A2 in the presence or absence of 75 pmol unlabeled competitor, irradiated, electrophoresed on a 15% SDS–polyacrylamide gel and autoradiographed. Telo1 (A) contains a single telomeric repeat and Telo3 (B) oligonucleotide has three repeats of the telomeric sequence [i.e. (TTAGGG)₃]. (C) Competition UV-cross-linking EMSAs performed by incubating ³²P end-labeled rA2RE11 and Telo1 with recombinant hnRNP A2 (10 pmol) in the presence or absence of unlabeled competitor, as above. The telomeric DNA repeat sequence competes with dA2RE11, but weakly with A2RE11, for the binding site on recombinant hnRNP A2.

Figure 4

A consensus oligonucleotide sequence for binding to hnRNP A2. (A) Point mutations were introduced into the dA2RE11 sequence at each position by replacement of the native nucleotide with the other three, separately generating all four oligodeoxyribonucleotides for each position. The ability of the modified oligonucleotides to compete with the unmutated dA2RE11 was assessed by using competition UV-cross-linking EMSAs with recombinant hnRNP A2 in the absence of heparin. Representative results are shown. Controls are shown on the left and the results for the mutations at the indicated positions of the A2RE11 are shown on the right. Competing oligodeoxynucleotides have the point mutations indicated above the autoradiographs. (B) The wild-type dA2RE11 sequence and single-nucleotide changes that still allow specific binding are shown along with the deduced consensus sequence. In deriving the consensus sequence it has been assumed that substitution at each position is without direct effect on the binding of the non-mutated nucleotides.

Figure 5

Both RRM domains of recombinant hnRNP A2 are required for binding the telomeric sequence. (A) Schematic representation of the recombinant proteins used. RRM, RNA recognition motif; GRD, glycine-rich domain. The prime indicates the presence of the additional 10 residues (179–189) beyond the C-terminal end of RRM2 that markedly increase the affinity of the concatenated RRMs for A2RE and A2RE11. (B) UV-cross-linking EMSA autoradiograph generated by incubating ³²P end-labeled Telo3 and equimolar concentrations of the recombinant proteins, irradiating the mixtures and separating the proteins on a 15% SDS–polyacrylamide gel. Free Telo3 probe is indicated at bottom left. Although the telomeric sequence does not appear to bind RRM1, it binds RRM2 and associates more tightly with the concatenated RRMs and the whole protein.

Figure 6

Recombinant hnRNP A2 protects telomeric DNA against DNase digestion. (A) Increasing concentrations of recombinant hnRNP A2 (1, 4 and 10 μM) were incubated with the ³²P end-labeled hexameric telomeric DNA repeat [Telo6, (TTAGGG)₆], or the complementary sequence [Anti6, (CCCTAA)₆], before adding the endonuclease DNase I. Breakdown products of the oligonucleotides were separated on a 20% polyacrylamide/7 M urea gel. Both oligonucleotides are extensively degraded in the absence of hnRNP A2. In the presence of this protein the telomeric sequence (left panel), but not the complementary sequence (right), is resistant to endonuclease digestion. (B) The GRD of hnRNP A2 is necessary but not sufficient for DNase I protection. Each recombinant protein (10 μM of RRM1, RRM2, RRM1+2′ or hnRNP A2) was assessed for its ability to protect the telomeric DNA. The proteins were incubated with ³²P end-labeled Telo6 or Anti6 before adding DNase I. Breakdown products of the oligonucleotides were separated on a 20% polyacrylamide/7 M urea gel and visualized by autoradiography. The individual or concatenated RRMs provided no protection: the intact protein was required, suggesting that the glycine-rich region is involved in protecting the telomeric sequence against enzymatic degradation. The telomeric sequence, but not its complement, is protected. (C) DNase I protection assay performed as described above, using 1, 4 and 10 μM of the recombinant GRD protein. This domain appears to be necessary but not sufficient for RNA protection.

Figure 7

hnRNP A2 binds the first 71 nt of human telomerase RNA. (A) ³²P-labeled hTR 5′ 71-nt was incubated with increasing amounts (0, 0.1, 0.5, 1, 2, 5 and 10 pmol) of recombinant hnRNP A2, and separated on a 15-cm 5% polyacrylamide gel. These assays were performed in the presence of 1 μg/μl heparin. The arrow on the right marks the retarded RNA–protein complex. (B) Assay was performed as above, except that the RNA used was first denatured by heating to 95°C for 5 min, cooled by placing immediately on ice and then incubated with protein. (C) ³²P-labeled hTR 5′ 71-nt RNA was incubated with 1, 10 and 100 μM of each recombinant protein, and separated on a 15-cm 5% native polyacrylamide gel. The single and double arrowheads point to the monomer and dimer forms of the RNA, respectively.

Figure 8

hnRNP A2/B1, PML and TRF2 are colocalized in nuclear bodies. Colocalization of hnRNP A2/B1, PML and hTRF2 at APBs in the nuclei of telomerase-negative, immortalized human fibroblasts (JFCF-6T.1J/1–4D cells). (A) hnRNP A2/B1 (FITC, green). (B) PML (Texas Red, red). (C) TRF2 (AMCA, blue). (D) Colocalization is indicated by the arrows in the merged image. Scale bar: 10 μm.