Figure 3. A specific element in the GFP coding region is sufficient for production of degradation intermediates by SOX.

(A) The modified GFP mRNA (GFPrep) contains an internal repeat of the first 201 basepairs of the GFP coding region (grey shaded area). (B) 293T cells were treated with control or Xrn1 siRNAs, then transfected with GFP or GFPrep +/− SOX. RNA was Northern blotted with a GFP 3′ UTR or an 18S probe. Arrowheads denote degradation intermediates. (C) Western blot shows the level of Xrn1 depletion in the experiment depicted in panel B, with actin as a loading control. (D) 5′ RACE was carried out on total RNA from 293T cells transfected with Xrn1 siRNAs and a GFP expression vector in the presence or absence of SOX. An internal GFP primer and a primer to the adapter ligated to the 5′ end of the RNAs were used to amplify GFP in each sample. Full length refers to the band corresponding to the intact GFP transcript, intermediate refers to the ∼300 bp band preferentially amplified in the lysate from SOX-expressing cells, and NT denotes the no template control. (E) A conserved TGAAG site located just upstream of the cleavage site was deleted from GFP to create GFP-ΔTGAAG. 293T cells were treated with control or Xrn1 siRNAs, then transfected with GFP or GFP ΔTGAAG in the presence or absence of SOX. RNA was Northern blotted with a GFP 3′ UTR or 18S probe (upper panels). Arrowheads denote degradation intermediates. Western blot (lower panels) shows the level of Xrn1 depletion in experiments depicted in panel E and F, with tubulin as a loading control. (F) Modified GFP reporters bearing an internal duplication of sequences around the GFP cleavage site were transfected in 293T cells treated with Xrn1 siRNAs +/− SOX. The inserted nucleotides are numbered based on their position relative to the GFP start codon. GFPrep (nt 1–201) is included as a positive control. RNA was Northern blotted with a GFP 3′ UTR or 18S probe. Arrowheads denote degradation intermediates.