ZFC3H1 depletion leads to cytoplasmic accumulation of endogenous 5′SS motif containing mRNAs (or intronic polyadenylated transcripts). (A) Workflow for RNA Frac-seq. ZFC3H1- or control-depleted U2OS cells were fractionated into nuclear and cytoplasmic/ER fractions (see Materials and Methods for more details). RNA was purified from these fractions and from total cell lysates, and then analyzed by Illumina sequencing. (B) Nuclear “N” and cytoplasmic “C” fractions were collected from ZFC3H1- or control-depleted U2OS cells, then separated by SDS-PAGE and analyzed by immunoblot for nuclear (Aly), ER (Trap-α), and cytoplasmic (tubulin) protein markers. (C) Lysates collected from ZFC3H1- or control-depleted U2OS cells (96 h post-transduction with lentiviral-delivered ZFC3H1-2 shRNA) were analyzed by immunoblot for ZFC3H1 and tubulin. (D) Fold change in total levels of intronic polyadenylated (IPA) transcripts (ZFC3H1 depletion vs. control depletion) (x-axis), plotted against the change in the total levels of fully processed mRNA (using cUTR reads, y-axis). Each dot corresponds to reads from one gene that is known to produce IPA transcripts (listed in Supplemental Table 1). Note that ZFC3H1 depletion leads to the up-regulation of IPA transcripts, but not fully processed mRNAs. (E) (Top) Schematic of a fully processed mRNA and IPA transcript generated from the same gene. Note that the IPA transcript is generated from a 3′ cleavage/polyadenylation signal in the first intron and contains a 5′SS motif. (Bottom) genome browser tracks of the PCF11 gene in control- “shCon” or ZFC3H1-depleted “shZFC” cells at 500× and 50× resolution. Note the large peak for the IPA transcript (intronic cleavage/polyadenylation site is denoted with a red arrow), which is up-regulated in ZFC3H1-depleted cells. Also note that the reads corresponding to the full-length transcript are unaffected by ZFC3H1 depletion. (F) Similar to D, except that the fold change of IPA transcript levels in the cytoplasmic fraction (ZFC3H1 depletion vs. control depletion, x-axis) is plotted against the fold change in the nuclear fraction (ZFC3H1 depletion vs. control depletion, y-axis). Note that ZFC3H1 depletion leads to cytoplasmic accumulation of many IPA transcripts (compare blue dots to red). To account for reads from fully processed mRNAs, the IPA transcript levels are normalized to the cUTR transcript levels of the same gene. (G) Similar to E, except the IPA peaks in the nuclear and cytoplasmic fractions are shown for PCF11. In control-depleted cells, reads from the PCF11 IPA are enriched in the nuclear, but not the cytoplasmic fractions. In ZFC3H1-depleted cells, reads from the PCF11 IPA are at comparable levels in the nuclear and cytoplasmic fractions. (H–J) Genome browser tracks of three IPA transcript-producing genes, “CCDC71,” “BRD3,” and “ZFPM1.” Note the accumulation of IPA transcripts in the cytoplasmic fractions upon ZFC3H1 depletion. The intronic 3′ cleavage/polyadenylation sites are denoted by red arrows.
