3’UTR-APA.

<p><b>(A)</b> Schematic of 3’UTR-APA that results in alternative 3’UTR isoforms. The region between two pAs, pA1 and pA2, is called alternative UTR (aUTR). AAA_n, poly(A) tail. CDS and 3’UTR are indicated. Regulation of APA is represented by RED (relative expression difference), whose formula is shown in the figure. #pA1 and #pA2 are numbers of poly(A) site-supporting (PASS) reads for pA1 and pA2, respectively. Test and Ctrl are test and control samples, respectively. <b>(B)</b> RED values for different samples. The top two most abundant APA isoforms based on the number of PASS reads of each gene were analyzed. Only genes with > = 20 PASS reads for proximal and distal pAs combined were used. The median RED value of each sample is shown as a thick black line and interquartile range (between the 25^th and 75^th percentiles) is indicated by a gray box. <b>(C)</b> An example of 3’UTR-APA regulation. Left, APA isoforms of <i>Timp2</i>. The gene structure and the zoomed-in 3’-most exon are shown on the top. Two pAs are indicated, and their 3’UTR lengths and RPM values in proliferating and differentiated C2C12 cells are shown. Sequence conservation of the shown region (based on mammals) is indicated, with the height of line reflecting the degree of conservation. RT-qPCR amplicons to study APA regulation are indicated. Middle, regulation of the two 3’UTR-APA isoforms of <i>Timp2</i> in several samples as indicated. RED values (distal pA vs. proximal pA, knockdown (KD) vs. siCtrl or differentiated cells vs. proliferating cells), q-values (Significance Analysis of Alternative Polyadenylation, SAAP, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005166#sec018" target="_blank">Materials and Methods</a> for detail), and direction of 3’UTR change (shortened, sh; or lengthened, Le) are indicated. Right, RT-qPCR analysis of 3’UTR-APA isoforms of <i>Timp2</i> in several samples. RT-qPCR ΔΔCycle threshold (Ct) values were calculated by comparing Ct difference between proximal and distal amplicons in test vs. ctrl samples. Test is a sample from knockdown or differentiated cells, and ctrl is a sample from siCtrl or proliferating cells. Note the samples used for RT-qPCR analysis were not the same as those used for 3’READS. Error bars are standard deviation based on two replicates. <b>(D)</b> Normalized number of genes with regulated 3’UTR-APA in each sample as examined by GAAP. Normalized number is based on (observed value—expected value), and zero is used if a value is negative. Red and blue bars represent genes with lengthened 3’UTRs (Le, distal pA isoform upregulated) and shortened 3’UTRs (Sh, proximal pA isoform upregulated), respectively. Q-value < 0.05 (SAAP) was used to select genes with significant 3’UTR-APA regulation. Only the top two most abundant APA isoforms (based on the number of PASS reads) of each gene were used for this analysis. Error bars are standard deviation based on 20 times of bootstrapping. Samples are sorted by the total number of genes with 3’UTR-APA changes. Data for C2C12 differentiation are shown at the bottom for comparison. Log2(Le/Sh) is log2(ratio) of the number of Le genes to the number of Sh genes. <b>(E)</b> Correlation between normalized no. of genes with 3’UTR-APA (Le+Sh in (D)) and percent of genes with >2 pAs regulated (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005166#pgen.1005166.s003" target="_blank">S3 Fig</a>). C2C12 differentiation data and siPABPN1 data are shown in red and others in gray. R² is based on linear regression of all dots. <b>(F)</b> Correlation between log2(Le/Sh) and median aUTR size change. Data from all samples shown in (D) except the siCFI-68 and siCF-25 samples are plotted, with C2C12 differentiation data and siPABPN1 data shown in red diamond and square, respectively, and others in gray dots. R² is based on linear regression of all gray dots. Median aUTR size change was calculated by the median aUTR size of lengthened 3’UTRs minus that of shortened 3’UTRs. Each gene was calculated once.