FIG. 4.

Lhp1p binds less efficiently to RNA substrates in the absence of Lsm proteins. (A to E) The strains GAL::HA-lsm3 (lanes 1 to 3), Lhp1p-ProtA (lanes 4 to 6), and Lhp1p-ProtA/GAL::HA-lsm3 (lanes 7 to 12) were grown at 30°C in RSG medium (lanes 1 to 9) or transferred to glucose medium for 24 h (lanes 10 to 12). Lysates were immunoprecipitated with IgG agarose, and RNA was recovered from the lysate (T), the immune supernatant (S), and the immunoprecipitate (P) and analyzed by Northern hybridization. Probe names are indicated in parentheses. RNA species are shown on the left. Due to the fact that depletion of Lsm proteins results in altered levels of some of the analyzed RNA species, different exposure times were used for different panels to adequately visualize RNAs. Approximately fourfold-more cell equivalents are loaded for the bound material. The arrowhead indicates the 5′ processed, 3′ unprocessed pre-tRNATyr. (F) Graphic representation of efficiency of immunoprecipitation by Lhp1p of RNAs from panels A to E in the Lhp1-ProtA/GAL::HA-lsm3 strain before (0 h) and after (8.5 and 24 h) depletion of Lsm3p. Values for each RNA species after depletion are expressed relative to the value before depletion, which is arbitrarily set at 1. Immunoprecipitation efficiency was calculated based on PhosphorImager quantification of Northern hybridization data from panels A to E. (G) Levels of Lsm3p, Lsm5p, and Lhp1p during Lsm depletion. Western blots of total protein were decorated with anti-HA to detect both HA-Lsm3p and HA-Lsm5p and with anti-protein A to detect Lhp1p-ProtA, following growth in galactose medium (0-h samples) or 24 h after transfer to glucose medium.