FIG. 3.

Components of the Lsm1p-7p/Pat1p/Dhh1p complex are required for efficient recruitment of BMV RNA3 to the site of replication. (A) Membrane association of RNA3. The wt and lsm1i yeast strains, expressing BMV RNA3 with and without 1a, were spheroplasted and lysed osmotically. The total lysate (T) was fractionated into a membrane-depleted supernatant (S) and a membrane-enriched pellet (P). The RNA3 level in each fraction was analyzed by Northern blotting. (B) Northern blot analysis of 1a-directed RNA3 accumulation in wt, lsm1Δ, lsm6Δ, lsm7Δ, pat1Δ, and dhh1Δ yeast strains. wt BMV RNA3 was transcribed from the yeast CUP1 promoter. 1a_A, ADH1 promoter-driven 1a expression; 1a_G, GAL1 promoter-driven 1a expression; −, no expression of 1a protein. Equal amounts of total yeast RNA were loaded in each lane. Northern blots were probed with a ³²P-labeled RNA that hybridizes with BMV (+)RNA3. Histograms show averages and standard errors of the means of relative RNA3 accumulation from three or more experiments. The average of RNA3 in wt yeast in the presence of 1a protein was set to 100.