Isoform-specific quantification of mRNA translational efficiency. (A) Polysome profile used to separate mRNAs into six fractions based on the number of bound ribosomes. Fractions are labeled by the average number of ribosomes bound per message. The two peaks in fraction 0 are the 60S and 80S ribosomal subunits. (B) Schematic of polysome-gradient sedimentation, illustrating calculation of translation rates for individual mRNA isoforms. In this example, the distal isoform has a greater average number of ribosomes bound (1.71 ribosomes/mRNA) compared to the proximal isoform (1.43 ribosomes/mRNA). Because isoforms share the same ORF, we did not normalize by ORF length when comparing values for tandem isoforms. (C) Polysome profiles for the DNA methyltransferase Dnmt1 mRNA, which has two tandem UTR isoforms colored red (proximal), and blue (distal). 2P-seq read counts were normalized to known amounts of yeast spike-in RNA. Translational efficiencies, calculated as the average number of ribosomes bound, are shown as vertical lines. (D) Comparison of translation rates for proximal and distal tandem 3′ UTR isoforms. Results for the 851 most highly expressed tandem 3′ UTR isoforms are shown in red, with results of the remainder shown in gray. Because tandem UTR isoforms presumably share the same ORF, translation rates were not divided by ORF length; doing so increased the correlation between proximal and distal isoforms but did not affect the tendency toward increased translation of the distal isoforms. (E) Translational efficiency differences for proximal versus distal tandem UTR isoforms with and without a 7-nt site for the let-7 miRNA in the distal region. Otherwise, as in Figure 4A.
