Protein interaction analysis links NRDE2 to pre-mRNA splicing. (A,B) Native TAP-MS analysis of 3A-NRDE2 (A) and 3A-NRDE2Δ200 (B) under low-salt conditions (100 mM NaCl) compared with untagged control and performed in three independent replicates for each sample. The red dashed line marks false discovery rate of 0.05. The components of the nuclear exosome are labeled in red. Proteins with GO term “RNA splicing” are depicted in green. Proteins highlighted in bold have also been identified in the yeast two-hybrid (Y2H) screen. Unlabeled gray dots represent significantly enriched ribosomal proteins. (C,D) Formaldehyde cross-linking TAP-MS analysis of 3A-NRDE2 (C) and 3A-NRDE2Δ200 (D) compared with untagged control and performed in three independent replicates for each sample. Same labeling scheme as in A and B applies. (E) GO term analysis of 87 high-confidence hits from a Y2H screen of full-length NRDE2 (see also Supplemental Table S2). (F) Streptavidin pull-down and coimmunoprecipitation of endogenous 3A-tagged proteins from cells transiently overexpressing V5-NRDE2 or V5-NRDE2Δ200 fused to 2A-Puro in the presence or absence of RNase A. (G–I) Live-cell spinning-disk microscopy of endogenous mNeonGreen-tagged NRDE2 and NRDE2Δ200 with endogenous mCherry-tagged NS marker U2AF2 (Chusainow et al. 2005). The imaging was performed in untreated cells (G), cells treated for 6 h with 1 µM splicing inhibitor Thailanstatin A (H), or cells treated for 2 h with 5 µg/mL transcription inhibitor Actinomycin D (I). Scale bar 10 µm.
