Figure 4.

PABPN1 is required for proper poly(A) tail length in muscle cells. Pure cultures of primary mouse myoblasts from limb (L), pharyngeal (Ph) and extraocular (EOM) muscles were transfected with PABPN1-2 (2) or control scrambled (C) siRNA. Cells were collected and total RNA was isolated. (A) Representative distribution of bulk poly(A) tails from different types of myoblasts following PABPN1 knockdown. Total RNA was labeled with [³²P] cytidine 3′,5′-bis(phosphate) using T4 RNA ligase and digested with RNase A/T1. Samples were resolved by electrophoresis in denaturing polyacrylamide gels and exposed to radiographic film. Cells treated with PABPN1 siRNA show a significant decrease in poly(A) tract length of ∼300 nt. (B) Densitometric quantification of poly(A) tracts of ∼300 nt as normalized to poly(A) tracts of ∼100 nt for control siRNA versus PABPN1-2 siRNA. A 50% decrease in the relative number of ∼300 nt poly(A) tails is observed following PABPN1 knockdown. Data are mean ± SD, *P < 0.05, n = 5. (C) Linker ligation-mediated poly(A) tail (LLM-PAT) assays were used to determine poly(A) tail length of specific transcripts following myoblast transfection with control or PABPN1-2 siRNA. mRNA transcripts of myogenin, MyoD, GAPDH and PGK1 showed a significant decrease in poly(A) tract length of ∼300 nt. Data are mean ± SD, *P < 0.05, n = 3.