Fig. 4

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