(A) Fluorescent images showing GFP-Dbp5 localization in strains carrying plasmid versions of GFP-DBP5, GFP-dbp5-L12A, GFP-dbp5-R423A, and GFP-dbp5-L12A/R423A. (B) Growth of integrated and untagged DBP5, dbp5-L12A, dbp5-R423A, and dbp5-L12A/R423A at 25°C, 30°C, and 37°C for 1 day. (C) FISH images showing poly(A)-RNA (gray) localization in integrated and untagged DBP5 and dbp5-L12A/R423A strains compared to the DAPi stained DNA mass (blue) before and after a 4 hr shift to 37°C. Scale bars = 2 μm. (D) Northern blot analysis of tRNAIleUAU (Probe1) in DBP5 (WT), mex67-5, dbp5-1, nup159-1, and gle1-4 strains before and after a 2 hr temperature shift to 37°C. The primary tRNA transcript is denoted as (P), and the end-matured intron-containing tRNA is denoted as (I). Ratio of mean integrated intensity between (I) and (P) species is measured and normalized to the wild type value. Ethidium bromide stained gel shows 5.8S and 5S rRNA species in bottom panel as a loading control. (E) Fluorescent images of plasmid expressed GFP-Dbp5L12A localization in DBP5, mex67-5, nup159-1, and gle1-4 strains before and after a 2 hr temperature shift to 37°C. Scale bars = 2 μm.