FIGURE 6.

Factors affecting YRA1 regulation in trans. (A) The THO complex is hypersensitive to overexpression of YRA1. Selected deletions of nonessential factors were transformed with a construct (pIA558) bearing wild-type, intron-containing YRA1 under the control of the GAL1 promoter or the empty vector. After growth of the transformants under noninducing conditions, equal amounts of cells were transferred onto synthetic medium containing galactose and lacking leucine to select for the presence of the plasmid. Cells were incubated for 3–4 d at the temperature indicated below the two panels. (B) YRA1 pre-mRNA levels are decreased in deletions of THO-complex subunits. The indicated strains were transformed with the same constructs as in A and grown in galactose-containing medium to early log-phase. Total RNA was applied to membrane and probed for the amount of total YRA1 (left panel), YRA1 pre-mRNA (middle panel), or a normalization control (U3 snRNA, right panel). Two serial dilutions are shown. (C) Yra1 protein levels are greatly elevated in deletions of THO-complex components. Protein extracts from wild-type and the indicated deletion strains were separated by electrophoresis on a 12% polyacrylamide/SDS gel and blotted to nitrocellulose, and the relative amounts of endogenous Yra1p were determined with a polyclonal αYra1p antibody. The positions of protein size markers (in kDa) are indicated on the right. Polyclonal antiserum against Brr3p/Gle1p was used to verify equal loading of the gel (lower panel). (D) Deletion of the cytoplasmic exonuclease Xrn1p overaccumulates YRA1 pre-mRNA approximately fivefold compared with wild type. Strains were analyzed for their endogenous pre-mRNA levels as in B. See text for more details.