Figure 4.

Nab2p Is Sequestered on Nuclear-Retained RNA in Export-Deficient Cells and Excess Nab2p Can Alleviate mRNA Downregulation

(A) SDS-PAGE of eluted samples from an RNA immunoprecipitation (IP) experiment in which endogenous Nab2p was immunoprecipitated from Mex67-AA cells without (“rapamycin –”) or treated with rapamycin for 30 min (“rapamycin +”). Before RNA IP, cells were not subjected to crosslinking (“0 J/cm²”), moderately cross-linked (“300 J/cm²,” ∼2 min cross-linking time), or strongly cross-linked (“700 J/cm²,” ∼5 min cross-linking time). Mock samples were negative control IPs using beads without anti-Nab2p antibody. Shown are IP eluates without (lanes 1–7) or with (lanes 8–14) prior RNaseA/T1 treatment. Top: western blotting analysis of Nab2p levels; bottom: autoradiogram of P³²-labeled SDS-PAGE gel (representative example of three independent repeats).

(B) Quantification of the P³²-labeled RNA signal from lanes 1–7 in (A). Background was estimated and subtracted on the basis of the mock sample. Single points represent values obtained from three individual repeats. Error bars represent SDs.

(C) Quantification of P³²-labeled RNA signal within the Nab2p contained band of lanes 8–14 from (A). Values were normalized as in Figure 4B. Error bars represent SDs from three independent repeats, and statistical significance was tested using a two-tailed paired t test.

(D) RT-qPCR analysis of selected 10 min 4tU-labeled RNAs purified from cells treated for 15 min with rapamycin as in Figure 3C. RNA series 2–4 show new RNA production from control Mex67-AA cells grown in glucose (series 3), which have been simultaneously depleted for Nab2p (Mex67-AA/Nab2-AA) (series 2) or containing a Nab2p expressing plasmid (Mex67-AA p2μ_NAB2) (series 4). Values derive from a minimum of four biological replicates, and error bars indicate SDs. Values for RNA series 3 were also included in the mean shown in Figure 3C. RNA series 5–9 show new RNAs from Mex67-AA cells containing an empty (series 5) or a pPgal_Nab2 (series 6–9) plasmid and incubated for 2.5, 5, 6, or 8 hr in galactose. Values are a mean of two biological replicates for the pPgal_Nab2 containing samples and an average of single replicates of all galactose incubation times for the empty plasmid sample. Error bars indicate median absolute deviation.

(E) Western blotting analysis of Nab2p levels in non-rapamycin-treated Mex67-AA cells transformed with either an empty 2μ pRS426 plasmid (−) or its counterpart (pNAB2) expressing wild-type Nab2p (+). Rpb3p served as a loading control.

(F) Western blotting analysis showing Nab2p and Rpb3p levels in cells containing a NAB2 gene under control of a galactose-inducible promoter (pPgal_Nab2) in glucose (GLU) condition and at the indicated times after galactose (GAL) addition. The image was edited to show only one of the repeats performed in parallel.

See also Figure S4.