Cleavage and polyadenylation of the β-globin transcripts.

(A) Detection of the downstream cleavage product (3′ fragment). The expression of Rat1 was silenced by RNAi in S2 cells expressing either mut or wt β-globin. Control cells were treated in parallel with GFP-dsRNA. Total RNA was purified and reverse-transcribed from dsRNA-treated cells, and the resulting cDNAs were quantified by qPCR with primers specific for the pre-mRNA, the mRNA (see Figure 4A) and the 3′ fragment (see the schematic picture above the histogram). The histogram shows the average values and standard deviations of β-globin levels, normalized to actin 5C RNA, from three independent experiments with two qPCR runs each (n = 6). The relative accumulation of downstream product was less pronounced in cells that expressed the mut β-globin gene than in cells that expressed the wt gene (p<0.01 using a paired, two-tailed Student's t-test, n = 6). (B) PCR-based detection of polyadenylated β-globin transcripts. Total RNA was purified from nuclear preparations of S2 cells expressing either mut or wt β-globin genes. The resulting cDNAs were used to amplify polyadenylated β-globin sequences by PCR with a 26 nt-long downstream primer complementary to the beginning of the poly(A) tail, as indicated in the figure (lanes 14). Control reactions with a 16 nt-long primer lacking the oligo(dT) extension were processed in parallel to rule out poly(A)-independent priming (lanes 58). Contamination with genomic DNA was assessed in parallel reactions without reverse transcriptase (RT-). (C) RT-qPCR quantification of polyadenylated β-globin transcripts. The samples described above were analyzed by RT-qPCR. Relative levels of polyadenylated β-globin transcripts, normalized to the total β-globin RNA, are shown. Values and standard deviations represent the average from two independent experiments with two qPCR runs each (n = 4). No significant differences were observed using a paired, two-tailed Student's t-test (p = 0.12, n = 4).