Polyadenylation-Dependent Control of Long Noncoding RNA Expression by the Poly(A)-Binding Protein Nuclear 1
Figure 5
PABPN1–dependent RNA decay is polyadenylation-dependent.
(A) Immunoprecipitation (IP) experiments showing that the SHG60 lncRNA is enriched in PABPN1 precipitates, but not in a control purification. The GAPDH mRNA and the SRP noncoding RNA were used as controls. (B) RNA enrichments (IP∶input ratio) in PABPN1 and control purifications were determined by real-time RT-PCR for the SHG60 RNA, the GAPDH mRNA, and the SRP RNA. Fold changes are relative to the control actin IP and normalized to the SRP RNA. The data and error bars represent the average and standard deviation from four independent experiments. (C) Quantitative RT-PCR analysis of RNA prepared from HeLa cells that were previously treated or not treated with cordycepin for 2 h. Fold increases are relative to untreated cells and normalized to the nonpolyadenylated SRP RNA. The data and error bars represent the average and standard deviation from three independent experiments. * p<0.05; Student's t-test. (D) Schematic diagram of the SHG60 constructs. White rectangles represent the noncoding exons, whereas the black box corresponds to the intronic SNORD60. The arrow indicates the position of the polyadenylation site as determined by 3′ RACE. The RBZ and H2A grey boxes correspond to the ribozyme and H2A terminator sequences, respectively. (E) HeLa cells treated with PABPN1–specific and control siRNAs were transfected with the control vector (lanes 1–4) or DNA constructs that express normal (lanes 5–8), ribozyme-processed (lanes 9–12), and H2A-processed (lanes 13–16) SHG60 lncRNA. Total RNA was treated with RNase H in the presence (+) or absence (−) of oligo(dT) before northern analysis using a SHG60-specific probe. The position of exogenous and endogenous SHG60 lncRNA is indicated on the right. The SRP RNA was used as a loading control. (F) The AAUAAA hexamer sequence of the SHG60 poly(A) signal is shown in bold with the two different hexamer variants used shown above the AAUAAA hexamer. The arrow shows the position of the SHG60 polyadenylation site, as determined by 3′ RACE. (G) HeLa cells were transfected with DNA constructs that express AAUAAA (lanes 1–2), AAGAAA (lanes 3–4), and AGUACU (lanes 5–6) SHG60 lncRNA. Endogenous and exogenous SHG60 lncRNA were detected in PABPN1–expressing conditions as shown in E (lanes 5–6).