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. 2011 Oct 27;7(10):e1002339. doi: 10.1371/journal.ppat.1002339

Figure 2. The 3′ degradation intermediate retains its poly(A) tail.

Figure 2

(A) 293T cells were transfected with GFP or a modified GFP reporter containing two copies of the flaviviral Xrn1-blocking element SLII within the GFP coding region, +/− SOX. A fraction of the RNA was treated with oligo(dT) and RNAse H to remove the poly(A) tail prior to Northern blotting with a GFP 3′ UTR or an 18S probe. Arrowhead denotes protected fragments. (B–C) 293T cells were transfected with control or Xrn1 siRNAs, followed by GFP (B) or DsRed2 (C) reporters +/− SOX. A fraction of the RNA was treated with oligo(dT) and RNAse H to remove the poly(A) tail prior to Northern blotting with a GFP 3′ UTR or an 18S probe. Xrn1 protein levels were assessed by Western blot (right panels in B and C). (D) 293T cells were treated with control siRNAs or siRNAs against the decapping complex protein Dcp1A. They were then transfected with GFP +/− SOX and/or the Dcp2 dominant negative mutant E148Q (Dcp2 DN). RNA was Northern blotted using a GFP 3′ UTR or an 18S probe. Western blots show the level of Dcp1A knockdown and Dcp2 DN overexpression. Actin serves as a loading control and grey lines indicate where intervening lanes have been cropped out. See also Figure S2.