Fig 2: Positioning of 75 bp sequence tags and quantitative changes following inhibition of cytoplasmic cap methylation. (A) The genome locations of the QuantSeq REV sequence tags are shown for one cytoplasmic capping target originally identified in (8) (VDAC3) and two non-target mRNA (STRN4, ACTB). A portion of the latter 75 bp sequence tag is shown in black with the AAUAAA poly(A) signal highlighted in red. (B) The QuantSeq REV data from Supplementary Table S2 are displayed by volcano plot, with transcripts that decrease with ?N-RNMT shown in red and those that increase shown on blue.

Fig 3: Differential expression of TOP mRNAs in response to inhibition of cytoplasmic cap methylation. (A) A heat map is shown for TOP mRNAs identified as changing in the five replicate libraries prepared after culturing in medium without (–Dox) or with (+Dox) doxycycline to induce ?N-RNMT expression. Ribosomal protein mRNAs are indicated on the right-hand side as RP, and non-ribosomal protein TOP mRNAs are indicated as TOP. The scale indicates z-scores for each determination. (B) Cytoplasmic RNA was recovered from cells carrying ?N-RNMT prior to addition of doxycycline to the medium and at 3 and 6 h after addition. Each preparation was spiked with an equal amount of mCherry mRNA and analyzed by RT-qPCR for 18S rRNA and XRCC6, RPS4X, RPL8, RPS3, EIF3D and EIF3K mRNA. The results were normalized to mCherry and are shown as the mean ± standard deviation (n = 3). *P < 0.05 by unpaired two-tailed t-test.

Fig 4: An inducible cell system to inactivate cytoplasmic cap methylation. A line of tetracycline-inducible U2OS cells was transfected with a plasmid bearing a tetracycline-regulated transgene expressing ?N-RNMT. Clonal lines were selected by growth in medium containing G418 and analyzed for potential leakiness and inducible expression. One clonal line was selected for use in this study. (A) Cells carrying the ?N-RNMT transgene were cultured for 24 h in the absence (–) or presence (+) of doxycycline (Dox), and cytoplasmic extracts were analyzed by western blotting with anti-RNMT antibody. The lower panel shows a portion of the same blot probed with antibody to EEF2 used in (7). (B) The same cell line grown on coverslips was again cultured for 24 h in the absence or presence of doxycycline, then fixed and stained with DAPI and antibody to the FLAG tag on ?N-RNMT. The areas in white boxes are shown enlarged at the bottom of the figure.