Fig 2: Depletion of ZC3H14 or THO components decreases steady-state mRNA levels. N2a cells were treated with siRNA to deplete THOC1, THOC5, or ZC3H14. Cells where either treated with individual siRNA or siRNA used in combination to deplete both THOC1 and ZC3H14 or both THOC5 and ZC3H14. Protein samples were analyzed by immunoblotting in (A) and RNA was analyzed by qRT-PCR in (B-D). (A) Antibodies against THOC1, THOC5, and ZC3H14 were used to confirm depletion of each protein with NPM1 serving as a loading control. Molecular weights are given in kDa and indicated to the left. Asterisk indicates a non-specific band recognized by the commercial THOC1 antibody. (B-D) RNA levels for (B) Atp5g1, (C) Psd95, and (D) Mapt mRNA are normalized to 18S transcript levels and are presented as fold change normalized to Scr control, with the value for the Scr control set to 1.0. Values represent the mean ± SEM for n = 3 independent experiments. All primers span exon-exon boundaries to specifically detect mature mRNA. Asterisks (*) indicate results that are statistically significant at *P < 0.05.

Fig 3: Knockdown of ZC3H14 and THO components cause extended bulk poly(A) tails. N2a cells were treated with scrambled (Scr), Thoc1, Thoc5 or Zc3h14 siRNA to deplete the corresponding proteins. (A) Immunoblot of protein samples from siRNA-treated N2a cells. Antibodies against THOC1, THOC5, and ZC3H14 were used to confirm depletion of each protein. eIF5 serves as a loading control for each sample. Molecular weights are given in kDa and indicated to the left. (B) Representative image of 3′-end labeled poly(A) tracts from bulk poly(A) tails of the siRNA-treated cells that have been resolved on a denaturing polyacrylamide gel and detected by autoradiography. (C) Line scan analysis of bulk poly(A) tail length. Poly(A) tails were quantified using ImageQuant software by plotting Pixel Intensity versus poly(A) tail length and normalizing to the start of the gel. Size markers are shown for 100 nt, 200 nt, and 300 nt. Line scan data are presented as means for three independent experiments. Statistical analysis was performed using a non-parametric, one-way ANOVA Kruskal-Wallis test. Asterisks (*) indicate results that are statistically significant at *P-value < 0.05.

Fig 4: ZC3H14 and THO components bind to common mRNA targets. (A) Endogenous ZC3H14, THOC5 and ALYREF RNA immunoprecipitation from N2a cells. The Input fractions and the Bound immunoprecipitated fractions were compared to the IgG immunoprecipitated control to confirm efficient and specific immunoprecipitation of ZC3H14, THOC5, and ALYREF. Molecular weights are indicated in kDa to the left. Asterisk indicates a non-specific band recognized by the commercial ALYREF antibody. (B–E) Quantification of RNA IP enrichment in (A). RNA isolated from the RNA-IPs was subjected to RT-qPCR analyses to detect the (B) Atp5g1, (C) Psd95, (D) Mapt and (E) Rplp0 transcripts. RNA levels in the bound fractions are normalized to input levels and are presented as fold change normalized to IgG control, with the value of the IgG control set to 1.0. Values represent the mean ± SEM for n = 3 independent experiments. Asterisks (*) indicate results that are statistical significance at *P-value < 0.05.

Fig 5: Immunoblots of GO Term-selected proteins show divergent phenotypes in isogenic COX17- and CTR1-null cells.A-C. Immunoblots of whole cell extracts from wild type (gray), two COX17 KO clonal lines (purple), and two CTR1 KO clonal lines (blue) probed for proteins involved in actin filaments or the cytoskeleton (MYLK, TALGN, ITGA1), components of the THO complex (THOC2, THOC5), the metalloreductase STEAP3, and secretory proteins (CTSB, CTSD, CHGB, and DBH; immature cathepsins are indicated with i). HSP90 was used as a loading control. Immunoblots were quantified by normalizing protein abundance to wild-type cells. The left column of images are from the same immunoblot and cropped for clarity. B. Quantification of COX17 KO protein abundance relative to wild type. Italicized numbers represent p values analyzed by unpaired mean difference two-sided permutation t-test. C. Quantification of COX17 and CTR1 KO protein abundance relative to wild-type cells. Italicized numbers represent p values analyzed by unpaired t-test with the following exceptions: MYLK and TGLN were analyzed by Fisher Exact Probability Test and CHGB was analyzed by Mann Whitney test. D. Immunofluorescence staining of WT and COX17 KO cells stained with the Golgi complex marker GM130 (green) and DAPI (blue) and quantification of Golgi area (pixels, each dot represents one cell). p value analyzed by unpaired t-test.