Fig 2: mTOR-coordinated cellular stress response network identified by IntMAP. (A) RNA- and PAS-Seq read alignments of Cebpg are shown. An absolute quantitation of long 3'-UTR and total (long+short 3'-UTR) Cebpg mRNAs was made using qPCR. The amounts of short versus long 3'-UTR transcripts are presented by copies/µg of total RNAs. The shortening index of Cebpg in the Tsc1-/- transcriptome was calculated based on absolute or normalized quantitation. qPCR results are from 4 technical replicates. Data represent the mean ± SEM. Two-tailed Student's t-tests were performed for statistical significance. (B) Western blotting for Cebpg, hnRNP A1 and Lamin A/C using WT and Tsc1-/- cell extracts. Relative amounts of Cebpg mRNA were measured by qPCR. qPCR results are from four technical replicates. Data represent the mean ± SEM. Two-tailed student t-tests were performed for statistical significance. *P = 7.97e–007. (C) Relative gene expression of Cebpg target genes in WT and Tsc1-/- MEFs was measured using qPCR. qPCR results are from 4 technical replicates. Data represent the mean ± SEM. Two-tailed Student's t-tests were performed for statistical significance. (D) RNAi knockdown (KD) of Cebpg in Tsc1-/- MEFs and the analysis of Cebpg target genes. Western blotting was conducted for Cebpg, Cpsf6, Tubulin, and hnRNP A1 using con or Cebpg KD Tsc1-/- cell extracts. Relative amounts of Cebpg mRNA in the KD cells compared to control cells were measured using qPCR. Expression of Cebpg target genes in the KD and control cells were quantitated using qPCR. qPCR results are from four technical replicates. Data represent the mean ± SEM. Two-tailed Student's t-tests were performed for statistical significance.

Fig 3: A regulatory role of 3'-UTR APA in Cebpg gene expression. (A) Schematic of proximal PAS knockout (KO) in Tsc1-/- MEFs. Using the CRISPR/Cas9-mediated genome editing tool, two juxtaposed proximal PASs in the 3'-UTR of Cebpg were deleted and Cebpg?PAS Tsc1-/- MEFs were established. (B) An absolute quantitation of total Cebpg mRNAs and Cebpg mRNA with long 3'-UTR was measured and presented by copies/µg total RNAs. The quantitation was made using three Cebpg?PAS Tsc1-/- MEF clones (#5, #11 and #12). The shortening index of Cebpg in these clones was calculated based on the absolute quantitation. qPCR results are from four technical replicates. Data represent the mean ± SEM. Two-tailed Student's t-tests were performed for statistical significance. (C) The RSI of selected genes in Cebpg?PAS#11 Tsc1-/- MEFs compared to control Tsc1-/- MEFs was measured using qPCR. qPCR results are from four technical replicates. Data represent the mean ± SEM. Two-tailed Student's t-tests were performed for statistical significance. (D) Cebpg?PAS Tsc1-/- MEFs were characterized using western blotting. Expression of C/EBP?, Cpsf6, Tubulin and hnRNP A1 protein was analyzed using total cell extracts from Cebpg?PAS and control Tsc1-/- MEFs. qPCR using the same set of cell lines was performed to measure the relative level of Cebpg mRNA. qPCR results are from 4 technical replicates. Data represent the mean ± SEM. Two-tailed Student's t-tests were performed for statistical significance. (E) Total and long 3'-UTR Cebpg mRNAs were analyzed using polysome profiling. Cytoplasmic extracts from Tsc1-/- and Cebpg?PAS Tsc1-/- MEFs were fractionated by 5–45% sucrose gradients. Percentage of total and long 3'-UTR Cebpg mRNAs was calculated in each fraction using qPCR. 80S monosome and polysomes in the fractionation were indicated. qPCR results are from four technical replicates. Data represent the mean ± SEM. (F) A serial deletion of the Cebpg 3'-UTR was tested for the regulation of Renilla luciferase expression. 100 (?0.1), 250 (?0.25), 350 (?0.35) and 500 (?0.5) nucleotide deletion constructs were tested. Renilla luciferase activity was normalized to firefly luciferase activity which was used as a transfection control. The assay was conducted four technical replicates. Three independent experiments showed similar results. One representative result is shown. Data represent the mean ± SEM. Two-tailed Student's t-tests were performed for statistical significance. P-values were obtained by comparing Short versus each deletion construct. *P < 0.00054. **P > 0.05. (G) Western blot analysis of multiple polyadenylation factors using WT and Tsc1-/- MEFs extracts is shown (left panel). RNAi-mediated knockdown of polyadenylation factors in WT MEFs were conducted (middle panel). The knockdown effect of those polyadenylation factors on the RSI was measured by qRT-PCR (right panel).