Structure-based Incorporation of 6-Methyl-8-(2-deoxy-β-ribofuranosyl)isoxanthopteridine into the Human Telomeric Repeat DNA as a Probe for UP1 Binding and Destabilization of G-tetrad Structures

Abstract

Heterogeneous ribonucleoprotein A1 (hnRNP A1) is an abundant nuclear protein that participates in RNA processing, alternative splicing, and chromosome maintenance. hnRNP A1 can be proteolyzed to unwinding protein (UP1), a 22.1-kDa protein that retains a high affinity for purine-rich single-stranded nucleic acids, including the human telomeric repeat (hTR) d(T-TAGGG) n . Using the structure of UP1 bound to hTR as a guide, we have incorporated the fluorescent guanine analog 6-MI at one of two positions within the DNA to facilitate binding studies. One is where 6-MI remains stacked with an adjacent purine, and another is where it becomes fully unstacked upon UP1 binding. The structures of both modified oligonucleotides complexed to UP1 were determined by x-ray crystallography to validate the efficacy of our design, and 6-MI has proven to be an excellent reporter molecule for single-stranded nucleic acid interactions in positions where there is a change in stacking environment upon complex formation. We have shown that UP1 affinity for d(TTAGGG) 2 is ϳ5 nM at 100 mM NaCl, pH 6.0, and our binding studies with d(TTAGG(6-MI)TTAGGG) show that binding is only modestly sensitive to salt and pH. UP1 also has a potent G-tetrad destabilizing activity that reduces the T m of the hTR sequence d(TAGGGT) 4 from 67.0°C to 36.1°C at physiological conditions (150 mM KCl, pH 7.0). Consistent with the structures determined by x-ray crystallography, UP1 is able to bind the hTR sequence in solution as a dimer and supports a model for hnRNP A1 binding to nucleic acids in arrays that may make a contiguous set of anti-parallel single-stranded nucleic acid binding clefts. These data suggest that seemingly disparate roles for hnRNP A1 in alternative splice site selection, RNA processing, RNA transport, and chromosome maintenance reflect its ability to bind a purine-rich consensus sequence (nYAGGn) and destabilize potentially deleterious G-tetrad structures.

hnRNP A1 associates with telomere ends and stimulates telomerase activity

“…This result is consistent with a previous study showing that UP1 can unfold a G-quadruplex formed by the G-rich strand of minisatellite repeats, d(GGCAG)n (Fukuda et al 2002). Shamoo and colleagues (Myers et al 2003) also reported that UP1 can reduce the T m of the human telomeric oligonucleotide d(TAGGGT) 4 in a 150 mM K + -containing solution, from 67.0°C to 36.1°C, consistent with a G-quadruplex-destabilizing activity of UP1. Although not proven, it seems likely that hnRNP A1 can also disrupt a G-G hairpin, as hnRNP A/B proteins are well known to have nucleic acid helix-destabilizing activity (Riva et al 1986).…”

Solution structure of the two RNA recognition motifs of hnRNP A1 using segmental isotope labeling: how the relative orientation between RRMs influences the nucleic acid binding topology

“…All these structures have been refined in the P4 3 2 1 2 space group from an identical tetragonal crystal form (pdb accession codes 2UP1, 1PGZ, 1PO6 and 1U1K to 1U1R) (Ding et al 1999;Myers et al 2003;Myers and Shamoo 2004). These bound structures are almost indistinguishable with a calculated average pairwise r.m.s.d.…”

High-affinity interaction of hnRNP A1 with conserved RNA structural elements is required for translation and replication of enterovirus 71

“…Thus, hnRNP A1 interacts with the SLII hairpin loop using a mode of recognition likely distinct from its established mode of binding single strand nucleic acids. 28,30,48 This observation suggests that hnRNP A1 can accommodate different RNA substrates using sequence and shape-specific recognition principles. The results obtained here are in good agreement with our previous study that showed UP1 binds the HIV-1 ESS3 hairpin loop as a highaffinity 1:1 complex; however, the sequence of the ESS3 apical loop (5'-GAUUAGU-3') more closely matches the hnRNP A1 "winner."…”