Structure of hnRNP D Complexed with Single-stranded Telomere DNA and Unfolding of the Quadruplex by Heterogeneous Nuclear Ribonucleoprotein D

Abstract

Heterogeneous nuclear ribonucleoprotein D, also known as AUF1, has two DNA/RNA-binding domains, each of which can specifically bind to single-stranded d(TTAGGG) n , the human telomeric repeat. Here, the structure of the C-terminal-binding domain (BD2) complexed with single-stranded d(TTAGGG) determined by NMR is presented. The structure has revealed that each residue of the d(TAG) segment is recognized by BD2 in a base-specific manner. The interactions deduced from the structure have been confirmed by gel retardation experiments with mutant BD2 and DNA. It is known that single-stranded DNA with the telomeric repeat tends to form a quadruplex and that the quadruplex has an inhibitory effect on telomere elongation by telomerase. This time it is revealed that BD2 unfolds the quadruplex of such DNA upon binding. Moreover, the effect of BD2 on the elongation by telomerase was examined in vitro. These results suggest the possible involvement of heterogeneous nuclear ribonucleoprotein D in maintenance of the telomere 3-overhang either through protection of a single-stranded DNA or destabilization of the potentially deleterious quadruplex structure for the elongation by telomerase. Heterogeneous nuclear ribonucleoprotein (hnRNP)1 D, also known as AUF1, was isolated from a HeLa cell nuclear extract as a protein that binds specifically to single-stranded d(T-TAGGG) n , the human telomeric DNA repeat (1). It also binds to r(UUAGGG) n (1) and the AU-rich element of the 3Ј-untranslated region of mRNA (2). It was shown biochemically that hnRNP D binding to the Gua-rich telomeric strand d(T-TAGGG) n destabilizes intrastrand Gua:Gua pairing and that hnRNP D interacts specifically with telomerase in human cell extracts (3). Thus, the involvement of hnRNP D in the maintenance of telomere DNA has been implied.hnRNP D consists of 306 amino acid residues and comprises two ribonucleoprotein (RNP)-type DNA/RNA-binding domains (BDs), BD1 (70 -173) and BD2 (174 -256), and a region rich in glycine and arginine residues (257-306) (4). The RNP-type BD is one of the most common eukaryotic protein sequence motifs for DNA/RNA binding (5), being found in hundreds of proteins (6 -9). A single BD of hnRNP D, either BD1 or BD2, is able to bind to DNA and RNA in a sequence-specific manner (4, 10, 11). The binding affinity of either BD1 or BD2 is comparable with that of the protein having both BD1 and BD2 (4). These results suggest that the basis of the interactions of hnRNP D with DNA and RNA can be addressed by examination with a single BD. We have already reported the structures of BD1 (10) and BD2 (11). We also qualitatively identified the surfaces of BD1 (10) and BD2 (11) interactive with DNA and RNA on chemical shift perturbation analysis, although it should be kept in mind that the perturbation is caused not only through the direct interaction but also thorough the indirect effect of the interaction. Essentially the same surfaces of the BDs are used for the interactions with DNA and RNA.Here, we present the structure of BD2 complexed with d(...

Human POT1 disrupts telomeric G-quadruplexes allowing telomerase extension in vitro

“…However, in marked contrast to the hPOT1 complex, the hnRNP-DNA complex is not a telomerase substrate (41,42). Thus, the telomere-specific hPOT1 protein appears to have an activity quite distinct from the activity of these more general nucleic-acid-binding proteins.…”

TERRA transcripts are bound by a complex array of RNA-binding proteins

“…4 ). As both hnRNP M and D / AUF1 have been previously shown to bind to single-stranded DNA 11,20,30 , absence of these two hnRNPs from telomeres may activate a mechanism that recruits TERRA to telomeres, possibly to increase telomere protection (see Fig. 5 for decreased telomere damage foci in hnRNP M and D / AUF knockdowns).…”

“…In particular, hnRNP A1, A2B1, A3, D and E can associate with the single-stranded telomeric repeats, although only hnRNP A1 has been shown to infl uence telomere length 11 -13 (reviewed in Ford et al 14 ). Th e hnRNP A1, D and C1 / C2 are also able to interact with human telomerase 12,15 -18 and hnRNP A1 and D can unwind G-quadruplexes 18,20 . Th e hnRNP interaction with TERRA transcripts and the eff ects on TERRA and telomeres that we described here further support a central role of hnRNPs in telomere biology.…”

“…Th e hnRNP proteins can also aff ect chromatin remodelling and transcription in a DNA-independent manner through protein -protein interactions (for example, hnRNP U binds RNA polymerase II) (reviewed in Carpenter et al 19 ). Th e hnRNP A1 and D have been also implicated in transcription owing to their ability to bind to and unwind G-quadruplexes 18,20 . A role for hnRNP A1 in transcription that does not involve unwinding of G-quadruplexes has been also reported 21 .…”

Structural basis of G-tract recognition and encaging by hnRNP F quasi-RRMs

“…2). The structures of hnRNP A1 and hnRNP D in complex with TAGGG DNA showed that these proteins contain classical RRMs, bind DNA sequences containing G-tracts using the canonical β-sheet surface and prevent G-quadruplex formation 46,49 show that, similarly to their effect on stem-loop RNA structures, hnRNP F qRRMs prevent the formation of G-quadruplex structures (Figs. 4a-c).…”