A novel method for poly(A) fractionation reveals a large population of mRNAs with a short poly(A) tail in mammalian cells

Abstract

The length of the poly(A) tail of an mRNA plays an important role in translational efficiency, mRNA stability and mRNA degradation. Regulated polyadenylation and deadenylation of specific mRNAs is involved in oogenesis, embryonic development, spermatogenesis, cell cycle progression and synaptic plasticity. Here we report a new technique to analyse the length of poly(A) tails and to separate a mixed population of mRNAs into fractions dependent on the length of their poly(A) tails. The method can be performed on crude lysate or total RNA, is fast, highly reproducible and minor changes in poly(A) tail length distribution are easily detected. We validated the method by analysing mRNAs known to undergo cytoplasmic polyadenylation during Xenopus laevis oocyte maturation. We then separated RNA from NIH3T3 cells into two fractions with short and long poly(A) tails and compared them by microarray analysis. In combination with the validation experiments, the results indicate that approximately 25% of the expressed genes have a poly(A) tail of less than 30 residues in a significant percentage of their transcripts.

Figure 1.

Poly(A) fractionation method. Schematic representation of the poly(A) fractionation protocol. Cells or total RNA are resuspended in a GTC buffer, mixed with a radiolabelled polyadenylated probe and biotinylated oligo(dT) and diluted. The oligo(dT) is then hybridized with the RNA, resulting in stacking of the oligonucleotides on longer poly(A) tails and bound to paramagnetic beads. The beads are washed with 0.5 × SSC and the RNA is eluted using decreasing salt concentrations, eluting the RNAs with short poly(A) tails first.

Figure 2.

Characterization of the binding and elution properties of biotinylated oligo(dT) chromatography. (A) Total RNA from NIH3T3 cells was subjected to oligo(dT) affinity chromatography. Starting material and the unbound fraction were examined for poly(A) tail lengths by 3′ end-labelling followed by RNase A and T1 digestion. The markers are the indicated sizes in number of nucleotides. (B) Total RNA from NIH3T3 cells was mixed with radiolabelled polyadenylated probe with A_0–500 and subjected to chromatography as above and eluted with 0.075 × SSC and water (Round 1). The unbound fraction was resubjected to the same procedure (Round 2). For total and unbound, equivalent amounts were loaded, while for the elutions, five times more was loaded. The marker is labelled with the number of nucleotides in excess over the unadenylated probe, which is 79 nt long. (C) Quantification of (B) for different lengths of poly(A) tails. The figure was quantified for each size using a phosphorimager and the background was subtracted. The numbers for the 0.075 × SSC and water elutions were divided by 5 to correct for the loading difference.

Figure 3.

Analysis of poly(A) tail length during Xenopus oogenesis. Stage 6 and mature X. laevis oocytes were lysed and mixed with radiolabelled polyadenylated probe with A_0–150. Both samples were subjected to oligo(dT) affinity chromatography and eluted using decreasing SSC concentrations. (A) Ten percent of the resulting fractions was analysed by urea–PAGE and the polyadenylated probe detected using a phosphor imager. Note that the water lane has only a weak signal in this instance because there is little probe with a poly(A) tail of more than 150 adenylate residues. (B) RT-PCR analysis of endogenous mRNAs in the fractions shown in (A). Samples in the no RT lane were treated exactly the same as the samples in the total RNA lane except for omitting the AMV-RT. (C) Northern analysis on 50% of the fractions shown in (A). Lanes with total RNA contain 15% of starting material. St 6, stage 6 oocytes; Mat, mature oocytes.

Figure 4.

Validation of poly(A) fractionation microarray. T, total RNA; U, unbound; S, short (oligoadenylated RNA eluted with 0.075× SSC); L, long (polyadenylated RNA eluted with H₂O). Gene names are according to Genbank, numbers in brackets indicate the corrected LogRatio obtained for the mRNA in the microarray screen. A high LogRatio should correlate with a short poly(A) tail. (A) Total RNA from NIH3T3 cells was mixed with radiolabelled polyadenylated probe and biotinylated oligo(dT) and then fractionated using the poly(A) fractionation method. Ten percent of the resulting fractions was analysed by urea–PAGE as in Figure 3A. The lane with total RNA contains 2% of starting material. The remainder of the fractions with short and long poly(A) tails was used for microarray analysis (see Supplementary Table 2). (B) Total RNA from NIH3T3 was subjected to RNaseH treatment in the presence of oligo(dT) (+ lanes) or used untreated (− lanes) and subjected to RL-PAT using specific sense primers (Supplementary Table 1) for genes identified in the microarray analysis. The size difference in PCR products between RNaseH treated and untreated corresponds to the length of the poly(A) tail. The numbers after the gene name indicate the corrected LogRatio obtained in the microarray experiment. (C) Total RNA from NIH3T3 cells was treated with RNaseH in the presence of oligo(dT) and an antisense oligo specific for Actb (+) or the presence of the specific oligo only (−). The sample treated with the specific oligo only was subsequently fractionated using the poly(A) fractionation method (U, S, L). Actb RNA was detected by Northern analysis. (D) RNA was fractionated as described in (A). RT-PCR was performed on the resulting fractions. Samples in the no RT lane (−) were treated the same as samples in the total RNA lane (T) except for the omission of Superscript III. (E) RNA was fractionated as described in (A). Twenty-five percent of each fraction was then analysed by northern blotting. Total: 5% of starting material. The numbers in brackets behind the gene names refer to the average corrected LogRatio (see Supplementary Table 2).