Fig. 3.
Analysis of the 3′ poly(A) ends of mRNAs with impaired binding to oligo(dT) or GST-4EK119A. (A) Schematic diagram of the RACE-PAT method used to directly determine the size of the 3′ poly(A) ends. This analysis was done for specific mRNAs, which were preferentially purified with either GST-4EK119A or oligo(dT). Total RNA was reverse transcribed with an oligo(dT) primer with a G/C rich anchor sequence [designated oligo(dT)12 primer]. Hybridization of the oligo(dT) primer to the mRNA 3′ poly(A) end occurs along the entire length of the 3′ poly(A) end. Different-sized cDNAs primed at all possible positions along with the poly(A) end will be synthesized after reverse transcription. Subsequent PCR amplification using this pool of cDNAs with a message specific primer (dotted box) and an oligo(dT)12 primer produces a mixture of PCR products, which include the length of the 3′ poly(A) end of the target mRNA. (B) Results of RACE-PAT for selected mRNAs. PCR-amplified products were analyzed by either 6% nondenaturing polyacrylamide (lanes 1–7) or 1% agarose gel electrophoresis (lanes 9–12). The predicted minimum size of the PCR products was compared with the actual size observed to obtain an estimate of the size of the 3′ poly(A) ends of a specific mRNA. For example, the minimum expected PCR product for the mRNA analyzed in lane 4 (replication protein A/14-kDa subunit, L07493) was 92 nt [62 + 30 nt oligo(dT)12-G/C anchor primer]. The actual size of the PCR product produced was 92 nt, indicating that this mRNA had a 3′ poly(A) end of 12 nt or less. The ethidium bromide-stained gels show a 100-bp DNA ladder (Invitrogen) in lanes 1 and 8. The estimated size for 3′ poly(A) ends of other mRNAs are shown: present only in GST-4EK119A purified mRNA (lane 2, H4 histone mRNA/X60484; lane 3, microsomal glutatione S-transferase 3 mRNA/AF026977; lane 4, replication protein A 14-kDa mRNA/L07493; see Table 3); mRNAs preferentially isolated by GST-4EK119A (lane 5, fau mRNA/X65923; lane 6, U6 snRNA-associated Sm-like protein/AA121509; lane 7, PROS-27 mRNA/X59417; see Table 2); and present only in oligo(dT)-purified mRNA (lane 9, pre-mRNA splicing factor PRP8/AB007510; lane 10, 1,4-α-glucan branching enzyme mRNA/L07956; lane 11, myeloid cell differentiation protein mRNA/L08246; lane 12, multidrug resistance-associated protein mRNA/AF085692; see Table 5). The short size of the 3′ poly(A) ends of the mRNAs in lanes 3–7 was also found by RACE-PAT analysis of RNA isolated from cultured Huh7 hepatoma cells (data not shown).