FIGURE 4.

(A) lsm1-9 and lsm1-14 alleles cause dominant inhibition of mRNA decay when overexpressed. Wild-type (FLAG-LSM1) cells transformed with 2μ vectors expressing different untagged alleles of LSM1 or an empty 2μ vector (indicated above the lanes in the top and bottom panels) were grown to log phase, after which RNA made from them was subjected to Northern analysis for MFA2pG mRNA (top) or lysates made from them were subjected to western analysis using anti-FLAG antibodies (bottom). The “% poly(G) fragment” values determined as described for Figure 3 and indicated at the bottom of the top panel are averages of at least two repeats. Positions of the MFA2pG mRNA and the poly(G) fragment are indicated on the right. In the bottom panel, lane 1 shows the western analysis of the lysate prepared from a wild-type strain that lacks a tag on Lsm1p. (B) Overexpression of the Lsm1p subunit results in an increase in the levels of the Lsm1-7–Pat1 complex. Lsm1-9p-containing complex purified from cells expressing lsm1-9 from a CEN or a 2μ vector (indicated above the lanes) was separated by SDS-PAGE and visualized by silver staining.