Figure 2.

Star-PAP regulates E6 mRNA levels by mediating the polyadenylation of the transcripts. (a) qRT–PCR¹⁵ demonstrated reduced production of the E6 messages after knockdown of Star-PAP in the presence or absence of VP-16 (50 μM, 6 h), and treatment with VP-16 alone also to some extent decreased E6 expression. TP53 and E6AP mRNA levels were not significantly affected by the treatments. Target mRNA abundance was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression (internal non-Star-PAP target control), and the mRNA expression levels displayed on charts were normalized to mock-treated control. Error bars represent s.e.m. of three independent experiments with triplicates for each experimental condition. Primers used for the analysis: HPV-18 E6 forward 5′-GCGACCCTACAAGCTACC TG-3′, reverse 5′-GTTGGAGTCGTTCCTGTCGT-3′; TP53 forward 5′-CCCGGACGATATTGAACAATGG-3′, reverse 5′-CAGAATGCAAGAAGCCCAGAC-3′; E6AP forward 5′-CCCTGATGATGTGTCTGTGG-3′, reverse 5′-GGCAAAGCCATTTCCAGATA-3′; GAPDH forward 5′-GAAGGTCGGAGTCAACGGATTT-3′, reverse 5′-GAATTTGCCATGGGTGGAAT-3′. (b) Detection of the intron-containing E6 expression by RT–PCR using primers within the first and second exons of E6, which could also amplify the spliced E6*I expression. (c) Poly(A) test (PAT) analysis of the poly(A) tail length of E6 mRNA in cells treated with Star-PAP knockdown or/and VP-16 (50 μM, 6 h). The length of the extended poly(A) reflects the polyadenylation potential of the PAP. Total RNA prepared with the TRIzol Reagent (Life Technologies, #15596-026, Grand Island, NY, USA) was subjected to annealing with phosphorylated oligo(dT)12–18 and ligation with oligo(dT)-anchor primers,²⁹ reverse transcribed and the E6 poly(A) mRNA generation assessed by PCR amplification of the corresponding complementary DNA using the gene-specific forward primer as described above and the oligo(dT)-anchor primer. The PCR products were then resolved on 1.5% agarose gel, stained with ethidium bromide and imaged.