Fig 2: Knockdown of cleavage and polyadenylation factors results in changes in isoform use and gene expression that overlap with quiescence. a Knockdown of cleavage and polyadenylation factors induces a shift in isoform expression. Real-time PCR was performed for the short and long isoforms of INF2 and BOC in proliferating fibroblasts expressing a control siRNA or an siRNA that targets CFIm25, CstF-64, or CPSF73. The short isoform of INF2 or BOC was significantly reduced in cells transfected with an siRNA against CstF64 or CPSF73. Plots show individual datapoints as dots. Bar graphs represent mean and average ± S.D. The number of replicates for control, CFIm25 and CPSF73 knockdown for short and long INF2 is 6. The number of replicates for CstF64 knockdown for short and long INF2 is 3. The number of replicates for all conditions for long BOC is 2, except the control, which had 3 replicates. The number of replicates for control and CFIm25 knockdown for short BOC is 3. The number of replicates for CstF64 and CPSF73 knockdown for short BOC is 2. Statistical significance in knockdown cells compared to control cells was determined for long and short isoforms with two-tailed, unpaired t-tests. b Overlap among genes that undergo APA with quiescence and knockdown of cleavage and polyadenylation factors. The overlap between genes that use the proximal polyadenylation site with quiescence and use a proximal polyadenylation site preferentially with CFIm25 knockdown is shown on the left. Overlap between genes that use distal polyadenylation sites with quiescence and genes that use distal polyadenylation sites with CPSF73 or CstF64 knockdown are shown in the middle and the right, respectively. c Overlap between genes upregulated with quiescence and genes upregulated with CstF-64 knockdown (left) and overlap between genes downregulated with quiescence and genes downregulated with CstF-64 knockdown (right). The overlap between groups of genes was tested using the hypergeometric test

Fig 3: Use of distal polyadenylation sites and lower levels of cleavage and polyadenylation factors during quiescence. a UCSC Genome browser views showing the long and short isoforms of INF2 and BOC. The differentially expressed exon is highlighted in cyan. b Real-time PCR validation of APA with quiescence. cDNA samples generated from fibroblasts that were proliferating, quiescent by contact inhibition or serum starvation, or induced into quiescence by serum-starvation and then restimulated, were analyzed with real-time PCR. Primers were designed to recognize the short (terminating at the proximal polyadenylation site) or long (terminating at the distal polyadenylation site) isoforms of INF2 or BOC. Transitioning fibroblasts into quiescence resulted in reduced expression of the short isoform of INF2 and increased expression of the long isoform of BOC. Restimulating quiescent fibroblasts resulted in expression patterns of the short and long isoforms that more closely resemble proliferating cells. Plots show individual datapoints as dots. Bar graphs represent mean and average ± S.D. The number of replicates for all conditions for short and long INF2 is 3. The number of replicates for all conditions for long BOC is 3. The number of replicates for P, 7dCI, and 7dCI-R for short BOC is 3. The number of replicates for 7dSS for short BOC is 2. Statistical significance in knockdown cells compared to control cells was determined for long and short isoforms with two-tailed, unpaired t tests. For all figures, one asterisk indicates p value < 0.05. Two asterisks indicate p value < 0.01. Three asterisks indicate p value < 0.001. c A shift toward expression of longer isoforms in quiescent fibroblasts. Proliferating and 7dCI were analyzed by polyadenylation site-enriched RNA-Seq. Relative use of the distal polyadenylation site (RUD) for individual genes in proliferating fibroblasts is plotted on the x-axis and RUD for the same gene in quiescent conditions is plotted on the y-axis. The dashed black line indicates y = x. The first plot (left) displays all genes with two detected polyadenylation sites. The middle plot displays UTR APA genes and the final plot (right) shows the same data for genes that undergo UR APA. d Immunoblotting was performed on protein lysates collected from proliferating, 7dCI and 7dSS fibroblasts for CstF-64, CFIm25, and CPSF73. Phosphorylation of serine 5 on RNA pol II CTD was monitored by immunoblotting and levels decline with quiescence. α-Tubulin was monitored as a loading control

Fig 4: Cleavage and polyadenylation factors are expressed at higher levels in fibroblasts near a wound than in fibroblasts of healthy skin. Mouse skin was collected 5 days after introduction of a punch biopsy. Normal mouse skin was collected 2 cm away from the wound. Samples were stained with immunohistochemistry for proliferation marker Ki-67, histone H4 as a control, or alternative polyadenylation and cleavage factors CstF-64, CPSF73 or CFIm25 (brown). Samples analyzed with immunohistochemistry were counterstained with hematoxylin (blue nuclei). Individual cells at different positions from the wounds were assigned positive or negative staining and the percentages are shown. Ki-67 does not label all dividing cells, and likely underestimates the fraction of cells that are actively cycling [122]. Levels of all three cleavage and polyadenylation factors were higher in the fibroblasts, myofibroblasts and immune cells proximal to a wound than in the fibroblast-rich dermal areas of healthy skin distal to the wound