RNA-seq and single-molecule FISH analysis of mRNA ER partitioning, abundance, and subcellular localization in HeLa Cas9 and SR KO cells. (A) Poly(A) mRNA fractional ER enrichments in HeLa Cas9 WT cells transfected with nontargeting siRNA versus HeLa Cas9 SRB KO2 cells transfected with SRA siRNA, 96 h post-transfection. Log2FC ≥ 1 is colored in blue for up-regulated, red in down-regulated. (B) Box/whisker plot depicting ER localization of mRNAs encoding cytosolic, secretory, single transmembrane (single-TM), and polytopic membrane (multi-TM) protein-encoding mRNAs in SR KO and control cell lines as in (A). (C) As in (A), but for total mRNA expression levels. (D) As in (B), but for total mRNA expression. See also Supplemental Data S2. (E) Representative single-molecule FISH (smFISH) imaging of subcellular mRNA distributions in HeLa Cas9, SRB KO HeLa (SRB KO), SRB KO transfected with nontargeting siRNA (SRB KO + nt siRNA), and SRB KO transfected with SRA-targeting siRNA. smFISH studies were performed for the cytosol-enriched mRNA GAPDH (green) and the ER-enriched mRNAs, GRP94 (red), GRP78 (red), and CD147 (red). Immunofluorescence staining of the ER-resident membrane protein TRAPα identifies the ER profile. Cell outlines were mapped from paired DIC images. Scale bar, 20 µm.
