Purifying mRNAs with a high-affinity eIF4E mutant identifies the short 3' poly(A) end phenotype

Abstract

The use of DNA microarrays has revolutionized the manner in which mRNA populations are analyzed. One limitation of the current technology is that mRNAs are often purified on the basis of their 3' poly(A) ends, which can be extremely short or absent in some mRNAs. To circumvent this limitation, we have developed a procedure for the purification of eukaryotic mRNAs using a mutant version of the mRNA 5' cap-binding protein (eIF4E) with increased affinity for the m7GTP moiety of the cap. By using this procedure, we have compared the populations of mammalian mRNAs purified by oligo(dT) and 5' cap selection with oligonucleotide microarrays. This analysis has identified a subpopulation of mRNAs that are present with short 3' poly(A) ends at steady state and are missed or underrepresented after purification by oligo(dT). These mRNAs may respond to specific posttranscriptional control mechanisms such as cytoplasmic polyadenylation.

Fig. 1.

(A) Relative ability of GST-4E_wild-type and GST-4E_K119A to bind 5′ capped mRNA. Batch mRNA-binding assays were performed to compare binding affinities of GST-4E_wild-type and GST-4E_K119A. 5′ capped ³²P-labeled mRNA was incubated in binding buffer with increasing amounts of GST-4E_wild-type and GST-4E_K119A bound to agarose beads (see Materials and Methods). The quantity of mRNA bound to GST-4E agarose beads was measured by Cerenkov counts. The estimated dissociation constants (K_d) of GST-4E_wild-type and GST-4E_K119A were 0.15 and 0.06 nM for capped mRNA, respectively. (B) Specificity of GST-4E_K119A for 5′ capped mRNA. The batch purification of mRNA using GST-4E_K119A was tested for its ability to bind both 5′ capped and uncapped mRNAs. Capped and uncapped mRNA synthesized in vitro using T7 polymerase were mixed with GST-4E_K119A agarose beads (50 μl), washed with 1× binding buffer, 500 μM GDP, and eluted with 1 mM m⁷GDP (see Materials and Methods). mRNA that remained bound to GST-4E beads despite the m⁷GDP elution step was recovered by extraction with acid phenol/chloroform. mRNA isolated by using GST-4E_K119A agarose beads are shown for purifications where 5′ capped (10–30%) and uncapped mRNA were used as starting material. mRNA present in each fraction was precipitated with ethanol and analyzed by 7 M urea/polyacrylamide (6%) gel electrophoresis and autoradiography. As determined by Cerenkov counts, the 1 mM m⁷GDP eluant (capped mRNA, lane 7) contained 85% of the total RNA recovered from the GST-4E_K119A beads. The arrow indicates the size of the mRNA (50 nt) used as starting material.

Fig. 2.

(A) Comparing 5′ cap- and 3′ poly(A)-dependent purifications of mRNA. Total RNA (315 μg) from normal liver was either mixed with GST-4E_K119A beads as described in Materials and Methods or applied to an oligo(dC₁₀T₃₀) column. The GST-4E_K119A matrix was washed, and mRNA was recovered by eluting with m⁷GDP (see Materials and Methods). An oligo(dT) column was used to purify mRNA as suggested by the manufacturer (Qiagen). Ten percent of the mRNA recovered from each purification was analyzed by formaldehyde agarose (1%) gel electrophoresis. An ethidium bromide-stained gel is shown: lane 1, 0.24- to 9.5-kb RNA ladder; lane 2, mRNA batch purified using GST-4E_K119A agarose beads; lane 3, mRNA purified using an oligo(dT) column. (B) In vitro translation of mRNA purified using GST-4E_K119A or oligo(dT). mRNA (1 μg) isolated by either GST-4E_K119A or oligo(dT) was translated in a nuclease-treated rabbit reticulocyte lysate with [³⁵S]methionine (see Materials and Methods). Protein products were analyzed by 10% SDS/PAGE and autoradiography: lane 1, control with no mRNA added; lane 2, mRNA purified with GST-4E_K119A; and lane 3, mRNA purified with oligo(dT). Molecular mass standards are shown.

Fig. 3.

Analysis of the 3′ poly(A) ends of mRNAs with impaired binding to oligo(dT) or GST-4E_K119A. (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-4E_K119A 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-4E_K119A 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-4E_K119A (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).