Long RBP10 mRNAs remain in the nucleus after DRBD18 depletion. Individual RBP10 mRNAs were detected by single-molecule fluorescent in situ hybridization with or without DRBD18 depletion for 12 or 24 h. In each case, 50 cells were analyzed. (A) Map showing the positions of the probes on the RBP10 mRNA, approximately to scale. The 3′-UTR divisions on the map correspond to those in Figure 2. The bars indicate the extent of mRNAs detected by the probe combinations. The dotted lines indicate that RNAs hybridizing to the different probe sets could have various lengths. The distances next to the bars indicate the position of the probe end on the mRNA. “Exp.”: Experiment. (B) Example image of RBP10 mRNAs in a wild-type cell, detected by single-molecule fluorescent in situ hybridization using probes 1, 2, and 4 (experiment I in panel A). DNA was stained with DAPI. The white arrow indicates an mRNA that hybridizes only to the coding region (open reading frame, ORF) and 3′ probe 2; the yellow arrow points to one of the four mRNAs that hybridize to probes 1, 2, and 4. (C) Mean numbers of long, medium, and short mRNAs per cell under different conditions. The data were from 50 cells. “None”: Cells with no RNAi plasmid; “-tet”: RNAi cells without tetracycline; and cells after 12 or 24 h tetracycline treatment. All slides were hybridized with the red, pink and green probes, and results for hybridization with the two different red probes are shown separately. “L3” indicates long mRNAs that hybridized with probes 1, 3, and 4 (experiment II), and “L2” indicates long mRNAs that hybridized with probes 1, 2, and 4 (experiment I). The black bars indicate mRNAs in the nucleus and the gray bars, mRNAs in the cytoplasm. Numbers for mRNAs hybridizing to only one probe, or red and green only, are in Supplemental Table S3. (D) Compilation of data from panel C, to allow comparison of the numbers of full-length and shorter mRNAs in the nucleus and cytoplasm. The key to symbols is on the right.
