Journal: Nucleic Acids Research

Article Title: Cytoplasmic poly-adenosine binding proteins modulate susceptibility of mRNAs to Pumilio-mediated decay

doi: 10.1093/nar/gkag075

Figure Lengend Snippet: PABPC1 overexpression stabilizes mRNAs, blocking PUM-mediated mRNA degradation. ( A ) Northern blot analysis of Nluc reporter mRNA containing either six wild-type PREs (6×PRE) or mutant PREs (6×PREmt) in HCT116 cells transfected with halotag (HT) or HT-PABPC1 expression plasmids. 18S rRNA served as a loading control, and ethidium bromide staining confirmed RNA integrity and loading. Three biological replicate samples are shown for each condition. As a marker for the Nluc mRNA with the poly(A) tail removed (A0), one RNA sample from the HT control was treated with oligo-dT15 and RNase H (RH + dT). Poly-adenylated (pA) and deadenylated (A0) Nluc species are indicated on the right. ( B ) Western blot analysis of effector protein expression. PABPC1 antibody detected both endogenous PABPC1 and overexpressed HT-PABPC1, while HT antibody confirmed the expression of HT. ( C ) Fold changes of either Nluc 6×PRE or Nluc 6×PREmt mRNA levels from panel (A) in response to overexpressed HT-PABPC1 were calculated relative to their respective HT negative controls. Data represent mean values ± SD, n = 3 biological replicates. ( D ) Fold changes of either Nluc 6×PRE mRNA levels from panel (A) in response to overexpressed HT-PABPC1 or the negative control HT were calculated relative to their respective or Nluc 6×PREmt negative controls. Data represent mean values ± SD, n = 3 biological replicates. For significance calling, * P < .05, ** P < .01, based on ordinary one-way ANOVA and Tukey test for multiple comparisons. ( E ) Tet-off transcription shut-off was performed to compare the half-lives of the Nluc 6×PRE and 6×PREmt reporter mRNAs in response to overexpressed HT-PABPC1 or negative control halotag (HT). A representative northern blot of Nluc reporters and the 18S ribosomal rRNA internal control is shown. Replicate blots are shown in . ( F ) Decay rates of Nluc 6×PRE and Nluc 6×PREmt in response to overexpression of HT-PABPC1, in comparison to HT. The fraction of each Nluc mRNA remaining, normalized to 18S rRNA, is plotted relative to time (hours) after inhibition of transcription. First-order exponential decay trend lines, calculated by non-linear regression analysis, are plotted for each experimental condition from three experimental replicates. n = 3; ± SD. On the right, mean Nluc mRNA half-lives from the experimental replicates are compared. n = 3; ± SD. For significance calling, * P < .05, ** P < .01, *** P < .001, based on ordinary one-way ANOVA and Tukey test for multiple comparisons. ( G ) A representative western blot confirming expression of HT-PABPC1 relative to endogenous PABPC1 using anti-PABPC1 antibody in each condition used for mRNA decay analysis. Western blot also confirmed expression of the HT protein. Histone H3 served as a loading control.

Article Snippet: For the Nluc mRNA marker with the poly(A) tail removed (A0), a control RNA sample was treated with oligo-dT15 (Promega) and RNase H (New England Biolabs) as previously described [ ].

Techniques: Over Expression, Blocking Assay, Northern Blot, Mutagenesis, Transfection, Expressing, Control, Staining, Marker, Western Blot, Negative Control, Comparison, Inhibition