Global analysis of differential polyadenylation of mRNAs in CPEB1 knockdown and CPEB4 knockdown at G2/M.

We compared the poly(A) tail length status of mRNAs of HeLa cells expressing a CPEB1 shRNA (CPEB1 knockdown) versus a control shRNA, and expressing a CPEB4 shRNA (CPEB4 knockdown) versus a control shRNA. Results provide insight into the extent of gene regulation mediated by CPEB1 and CPEB4 activity during mitotic cell cycle progression. The different shRNA expressing cells were synchronized with double thymidine blockade (12 hours with 2 mM thymidine, 12 hours release, and 12 hours with 2 mM thymidine), and samples were taken after 8 hours release (G2/M phase). For each shRNA expressing HeLa cell line total RNA was purified by two different procedures: poly(U) chromatography and oligo(dT)-chromatography. Poly(U)-chromatography (Jacobson, 1987): 100 μg of total RNA were bound to poly(U)-sepharose (Sigma) and eluted at 35ºC to isolate mRNAs with short poly(A) tail (<30As, SHORT fraction). Oligo(dT) chromatography: mRNAs were purified independently of their poly(A) tail length with Ambion Poly(A)Purist kit from 20 μg total RNA (ALL fraction). Jacobson, A. Purification and fractionation of poly(A)+ RNA. Methods in Enzymology (1987) 152: 254-261. Keywords: knock-down experiment Comparison of ALL fraction mRNAs and SHORT fraction mRNAs of cells expressing control shRNA, or CPEB1 shRNA, or CPEB4 shRNA measured after 8 hours release (G2/M) from double thymidine blockade; hybridisations are then used for comparison between control shRNA expressing cells and each of the two CPEB1 shRNA or CPEB4 shRNA expressing cells, as well as, comparison between the two knockdowns; 3 biological replicates for each shRNA expressing cells; two technical replicates with dye swapping per comparison.