Fig. 2

Sgf73p functions in mRNP quality control. a Spotting assays to assess genetic interactions between rrp6Δ and SAGA subunit-deletion mutants. Cells were spotted onto YPD plates with five-fold serial dilutions and incubated at 30 and 37 °C. b Co-IP assay of Sgf73p (α-MYC) and Rrp6p (α-FLAG). Rrp6p (α-5xFLAG) was immobilized on anti-FLAG M2 affinity gel and co-IP was assessed by Western blotting. c (Left panel) FISH analyses of wildtype, rrp6Δ, sgf73Δ, and sgf73Δrrp6Δ cells. Poly-(A) + RNA was detected using Cy3-labeled oligo(dT) probes and DNA was counterstained with DAPI. Scale bar, 5 μm. (Right panel) Quantification graph of poly-(A) + RNA intensity. Poly-(A) + RNA intensity was quantified using the same scan width of respective nuclei (determined by DAPI staining). d Spotting assays to assess the genetic interaction between rna14-3 and sgf73Δ. Cells were spotted onto YPD plates with five-fold serial dilutions and incubated at 25 and 32 °C. e Quantification of read-through transcripts from the HSP104 gene in wildtype and sgf73Δ cells. Wildtype and sgf73Δ cells were depleted of Rna14p using the anchor-away method (+Rap). Levels of read-through transcripts were normalized by 3′-transcripts. Standard deviations of three independent experiments are shown by error bars and P-values were determined by Student’s t-test (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001). WCE, whole cell extract. RT, read-through