(A)(i) Poly(A)-ClickSeq (PAC-seq) track showing reduced NUDT21 and (ii-iv) PAC-seq tracks showing altered alternative polyadenylation in example genes MECP2, VMA21, and PAK1. n = 3/treatment. Peaks are 3′ end sequencing reads. Multiple peaks in a gene indicate multiple mRNA isoforms with different cleavage and polyadenylation sites. Bracketed numbers show counts per million; kb stands for kilobases. (B) Schemata of the mRNA isoforms identified by PAC-seq for MECP2, VMA21, and PAK1 (top). Triangles show the binding sites of the qPCR primers used to validate the PAC-seq results. The gray primers detect all mRNA isoforms for the target gene, whereas the green primers only detect the isoform with the longest 3′ UTR. Gapdh-normalized RT-qPCR quantification showing relatively less of the long isoforms in the shNUDT21-infected neurons (bottom) for MECP2, p=0.0007 (i), VMA21, p=0.01 (ii), and PAK1, p=0.005 (iii). Error bars indicate SEM. Data analyzed by unpaired, two-tailed t-test. (C) Volcano plot showing relative mRNA length change in shNUDT21-infected neurons compared to shScramble-infected controls. The horizontal, dashed line shows Padjusted = 0.05, n = 3/treatment. NUDT21 loss in neurons predominantly results in shorter mRNAs (p<0.0001, two-tailed chi-square test). >90% of reads are in 3′ UTRs (Figure 6—figure supplement 1A). Principle component analysis (PCA) shows sample separation by treatment (Figure 6—figure supplement 1B). Distal cleavage sites of NUDT21-regulated mRNAs are enriched for the CFIm25 binding motif, UGUA, in mRNAs that shorten after NUDT21 knockdown, but not in non-target mRNAs (Figure 6—figure supplement 1C). (E) Mass spectrometry quantification of protein level fold change for genes with significantly altered APA (Padjusted <0.05). mRNA shortening predominantly results in increased protein levels (p<0.0001, two-tailed conditional chi-square test). Source files for the PAC-seq and mass spectrometry quantification data are available in Figure 6—source data 1. *p<0.05; **p<0.01; ***p<0.001.