The size of the 3′ decay intermediate is independent of the stop codon. Three variants of the Hsp70 reporter construct carrying the translation stop codon at different locations were used. The position of the stop codon is indicated on top: wild-type position (0), 39 nt and 66 nt upstream. A reporter variant with a 22 nt insertion immediately downstream from the stop codon was also used. S2 cells stably transformed with the constructs were treated with the Dcp2 dsRNA. Transcription of the reporter was induced by addition of 0.5 mM CuSO4. After 1 h, total RNA was isolated and analyzed by Northern blot. DNA probes were directed against reporter regions 1–51 (0+22) and 350–400 (0, −39 and −66). Because of the unexpected result, reporter mRNA was amplified from the experimental samples and the positions of the stop codons were confirmed by sequencing.
