Newly exported ribosomes and mRNAs localize to the ER in the absence of SR expression. (A,B) Representative time course of ribosomal subunit synthesis, nuclear export, and trafficking between the cytosol and ER. HeLa Cas9 WT cells transfected with nontargeting siRNA and HeLa Cas9 SRB KO2 cells transfected with SRA siRNA were pulse-labeled with 4SU 96 h post-transfection for 15 min and chased with excess uridine for the indicated time points. Cells were fractionated into cytosol and ER fractions, RNA was extracted, 4SU-RNA biotinylated, and RNA fractions were separated by denaturing agarose gel electrophoresis. Total RNA was detected by methylene blue (A) and 4SU-labeled RNA was detected by streptavidin-IRDye 800 imaging (B) of the same transfer membrane. (C) Quantitation of 4U 28S and 18S rRNA bands from (B). Data are averages of band intensity from two biological replicate RNA gels ± SD. (D) Time course of 4SU-poly(A) mRNA accumulation in the cytosol and ER fractions in HeLa cells, with the total representing the sum of the two fractions. (E) Fractional enrichments of newly exported, 4SU-poly(A) cytoplasmic, secretory, single transmembrane (single-TM), and polytopic membrane (multi-TM) protein-encoding mRNAs in the ER fraction. (F) As in (E), with cells treated with harringtonine at the start of the 4SU pulse-labeling period. (G) Comparison of 4SU-poly(A) mRNA enrichments for cytoplasmic, secretory, single transmembrane (single-TM), and polytopic membrane (multi-TM) protein-encoding mRNAs between HeLa Cas9 WT cells transfected with nontargeting siRNA and HeLa Cas9 SRB cells transfected with SRA siRNA 96 h post-transfection.
