Figure 2.

Puf6 associates with non-polysomal ASH1 mRNA. A, schematic representation of the modified endogenous ASH1 gene. HA tag was fused to the N terminus of Ash1. Six repeats of MS2 stem loops were fused to the 3′-UTR of ASH1 mRNA. B, extracts of yeast cells were fractionated in 10–50% linear sucrose gradients. An A₂₅₄ plot corresponding to the polysomal fractions is shown. C, total RNAs were isolated from the input and sucrose fractions. An equal amount of RNA from each fraction was subjected to Northern blotting to detect the distribution of ASH1 mRNA in the sucrose gradient (arrows) with GAPDH mRNA as an internal control. D, equal amounts of protein from each fraction were subjected to Western blotting for detecting the expression of Puf6. Tubulin was used as an internal control. E, ASH1-MS2 mRNA was precipitated using recombinant MBP-MCP (MBP binds to amylose resins), as shown in the top panel. Fractions 3 and 7 are non-polysomal and polysomal fractions. Puf6 and She2 were detected in the input and the precipitates of the non-polysomal fraction. Dcp1 and Pgk1 were used as negative controls.