FIGURE 4:

Elevated levels of tRNA and rRNA fragments in Rnt2 double-KO strains. (A) Southern blot analysis confirming complete replacement of a second RNT2 locus with the bsr2 resistance cassette. Genomic DNA from wild-type (wt) cells and three independent double-targeted clonal cell lines was digested with appropriate restriction enzymes to resolve wild-type and KO chromosomes as schematized above each blot. (B) RNA harvested from growing cells or cells starved for 3.5 h from two independent cell lines for each Rnt2 double KO, stained with SYBR Gold (left). Results are shown from Northern blot hybridization to detect 5′ and 3′ fragments of tRNA Gly and tRNA Ala (right). (C) Northern blot hybridization analysis of RNA as earlier described. Results from probes complementary to 5S rRNA, 5.8S rRNA, SRP RNA, small nuclear RNA U6, and small nucleolar RNATtnuCD32 are shown.