Figure 1: Design and biochemical characterization of ASREs.

(a) Structures of the PUF domain of human pumilio1 (PDB ID: 1M8W) bound by NRE-19 RNA target (left) and the PIN domain of Smg6 (right; PDB ID: 2HWW). Three Asp residues (indicated in green) near PIN-active site were marked. (b) ASRE with PUF–PIN configuration was incubated with a cognate RNA substrate for 30 min, and the products were resolved in urea–PAGE gel. Inverted ASRE (PIN–PUF) was also tested with the same reaction condition (lane 5 and 6). (c) Time course of RNA digestion by ASRE(6-2/7-2). Digestion was followed in the standard reaction buffer containing 3 mM Mn²⁺. (d) RNA substrates containing either an NRE site (5′-UGUAUAUA-3′) or 7u6g site (5′-UugAUAUA-3′) were incubated with ASRE(wt) or ASRE(6-2/7-2). The cognate target–ASRE pairs were indicated with same colour. Lanes 1 and 4 are controls without enzyme. (e) The RNA cleavage activities of ASREs are dependent on different divalent metal ions. In all lanes, the concentrations of metal ions were 3 mM. (f) ASRE cleavage site was mapped by 5′- and 3′-RACE using gel-purified RNA products. The positions of two cleavage sites are indicated with arrows and the relative frequencies are plotted. The 8-nt binding sequence of PUF is shown in bold. (g) The 'curve back' model of ASRE best explains the cleavage positions mapped in f.