Fig 2: Depletion of ZC3H14 or THOC1 causes accumulation of target pre-mRNA in the cytoplasm. N2a cells were treated with scrambled (Scr), Thoc1, or Zc3h14 siRNA and fractionated into Total, Nuclear, and Cytoplasmic (Cyto) fractions. Protein and RNA from these fractions were analyzed by immunoblotting (A) and qRT-PCR (C-F). (A) Fractions were probed with antibodies to detect ZC3H14 and THOC1 to confirm efficient depletion. Antibodies against the nuclear protein NUP62 and the cytoplasmic protein HSP90 were used to normalize loading and confirm proper fractionation. Molecular weights are given in kDa and indicated to the left. Asterisks indicate non-specific bands recognized by the commercial THOC1 and NUP62 antibody. (B) Schematic of pre-mRNA primers. The locations of the qPCR primers are indicated by arrows in the schematics above the targeted regions. Exons are represented as boxes and introns are represented as lines for each pre-mRNA. Dashed lines indicate regions of the transcript that are not shown. (C–F) Quantification of the total, nuclear, and cytoplasmic (Cyto) pre-mRNA levels in ZC3H14 or THOC1 depleted samples compared to Scr control treated samples. Pre-mRNA levels for (C) Atp5g1, (D) Psd95, (E) Mapt and (F) Rplp0 pre-mRNA were normalized to 18S transcript levels and are presented as fold change normalized to Scr control. Experimental groups for each fraction (total, nuclear, and cytoplasmic) were compared to the Scr control (which was set to 1.0) within each fraction to assess statistically significant differences. Values represent the mean ± SEM for n = 3 independent experiments. Asterisks (*) indicate results that are statistically significant at *P-value < 0.05.

Fig 3: Loss of ZC3H14 affects NXF1 target mRNA selection. Endogenous NXF1 was immunoprecipitated from N2a cells treated with scrambled (Scr), Thoc1 or Zc3h14 siRNA. (A) Immunoblot of NXF1 immunoprecipitation. Antibodies against THOC1 and ZC3H14 were used to confirm knockdown of ZC3H14 and THOC1. NPM1 serves as a loading control. Molecular weights are given in kDa and indicated on the left. Asterisk indicates a non-specific band recognized by the commercial THOC1 antibody. (B–E) Quantification of RNA-IP enrichment. RNA isolated from the NXF1 RNA-IP was subjected to qRT-PCR analyzed to detect (B) Atp5g1, (C) Psd95, (D) Mapt and (E) Rplp0 pre-mRNA. RNA levels in the NXF1 bound fractions are normalized to respective input levels and are presented as fold change normalized to Scr control, which was set to 1.0. The locations of the qPCR primers are indicated by arrows in the schematic above the graph. Exons are represented as boxes and introns are represented as lines for each pre-mRNA. Dashed lines indicate regions of the transcript that are not shown. Values represent the mean ± SEM for n = 3 independent experiments. Asterisks (*) indicate that the data are statistically significant at *P-value < 0.05.

Fig 4: ZC3H14 interacts with the THO complex. (A) Co-immunoprecipitation of ZC3H14 and THO components from mouse brain lysate. The Input and immunoprecipitated samples for ZC3H14, ZC3H14 + RNase, and control IgG are shown. THOC1 and THOC2 serve as representative members of the THO complex, together with the THO-associated protein ALYREF. HuR serves as a negative control. Asterisks indicate non-specific protein bands detected by the commercial antibodies. (B) Reciprocal co-immunoprecipitation using anti-THOC1. The Input and immunoprecipitated samples for THOC1, THOC1 + RNase and control IgG immunoprecipitations were probed to detect THOC1, ZC3H14 and THOC2 proteins. (C) Sedimentation of ZC3H14 protein complexes from the nuclear fraction of mouse brain lysate through a 10–50% glycerol gradient. Gradient fractions from top to bottom were resolved on SDS-PAGE from left to right. Fractions were probed with antibodies to detect ZC3H14, THOC1, THOC2, ALYREF and Nucleophosmin (NPM1) as a control. The Input sample is shown on the left. Overlapping fractions are outlined. Molecular weights are shown in kDa and indicated to the left.

Fig 5: ZC3H14 interacts with multiple mRNA processing proteins in the brain. (A) Fractionation of mouse brain lysate. Equal amounts of protein lysate from Total, Nuclear, or Cytoplasmic (Cyto) fractions were analyzed by immunoblotting with ZC3H14 antibody. The ZC3H14 antibody recognizes multiple isoforms of ZC3H14 resulting from alternative splicing of the ZC3H14 gene (15). To ensure proper fractionation, a nuclear marker (NUP93) and a cytoplasmic marker (eIF5) are shown. Molecular weights are indicated in kDa. (B) The gene ontology (GO) terms for the proteins that are mostly highly enriched within the immunoprecipitation of ZC3H14 compared to IgG control as identified by tandem mass spectrometry. These proteins are grouped based on the three GO domains: Biological Process, Cellular Component, and Molecular Function. The inclusion criteria for this analysis were a Z-score of > 4.5, a P-value of < 0.05, and at least three gene terms per GO term. (C) The gene terms that cluster into the listed GO terms in (B) are depicted. Colored lines connect GO categories to GO terms and individual term members. All core members of the THO complex (THOC1, THOC2, THOC3, THOC5, THOC6, THOC7) are present (indicated by an asterisk) as well as the THO associated proteins CHTOP and ALYREF (underlined).