The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element

Abstract

AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5 ′ -NNCCAC-3 ′ and 5A or C; N and N ′ form Watson-Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences.

The structure, function and evolution of proteins that bind DNA and RNA

“…The dual nucleic acid binding activity of NF-κB had been demonstrated in vitro many years prior to the discovery of an endogenous RNA target 10 , suggesting that transcription factors that are DRBPs such as AML1/RUNX1, whose RNA binding capacity has only been shown in vitro 19 , may have also endogenous RNA targets awaiting discovery. Although structural information on the interaction of human proteins with their RNA decoys is lacking, a recent study demonstrates an elaborate mechanism of an analogous bacterial system, the sequestration of RsmE by the ncRNA RsmZ 11 .…”

“…Not only does this indicate that proteins with evolutionary conserved DNA-binding activities are capable of binding RNA (and vice-versa), but also that some nucleic acid substrates may be similar enough in sequence and structure to foment protein promiscuity. As mentioned above, this phenomenon is exploited by RNAs, both endogenous and artificial, that function as decoys in order to modulate DRBP function 1,19,136 .…”

Characterisation of an aptamer against the Runt domain of AML1 (RUNX1) by NMR and mutational analyses

“…The unimportant residues are coloured grey. (B) Effect of mutation on Apt1‐S as reported previously . The importance of residues is represented by their colouring, the same as in panel A.…”

“…Expression and purification of AML1 RD and its mutants AML1 N-terminal fragment (amino acids 1-188, referred to as the RD) and its mutants were prepared as described previously [21,22]. The purified proteins were dialysed against buffer [20 mM sodium phosphate (pH 6.5), 2 mM magnesium acetate, 300 mM potassium acetate, 50% glycerol and 1 mM DTT] and stored at À25°C.…”

“…RNA aptamers that bind to RD were previously studied regarding their potential utility in the diagnosis and treatment of AML1-related diseases [20][21][22][23][24]. We have already obtained RNA aptamers (Apt1-S and S4-S) that show higher affinity (K d = 0.99 AE 0.02 nM and 0.034 AE 0.004 nM, respectively) than the Runt-binding double-stranded DNA element (RDE, K d = 9.6 AE 0.2 nM) [21,22]. Structural study of Apt1-S using NMR revealed that it contains a DNA-mimicking motif, which adopts a B-type DNA-like conformation [23,25].…”